Methods, Compounds and Compositions for Modulating Blood Brain Barrier Integrity and Re-myelination

ABSTRACT

Disclosed herein are methods and compositions for increasing and decreasing the permeability of the blood brain barrier for the treatment of diseases and conditions and to facilitate the delivery of agents to the brain, as well as methods and compositions for promoting re-myelination and preventing de-myelination. Compositions include RGMa, soluble RGMa, and functional fragments and variants thereof, RGMc, soluble RGMc, and functional fragments and variants thereof, and Neogenin peptides including 4Ig.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/762,497 filedMar. 22, 2018, which is a national phase application under 35 U.S.C. §371 of International Application No. PCT/CA2016/051122 filed Sep. 23,2016, which claims the benefit of U.S. Provisional Patent ApplicationSer. No. 62/222,697, filed Sep. 23, 2015, and of U.S. Provisional PatentApplication Ser. No. 62/338,793, filed May 19, 2016, the entire contentsof each of which are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING

The present application includes as part of its description a sequencelisting that includes 6 sequences and which was filed with thisapplication in electronic form and this sequence listing is incorporatedinto the present application in its entirety.

TECHNICAL FIELD

The present invention relates to methods of modulating blood brainbarrier (BBB) integrity, and methods of treating a disease in which BBBdisruption are detrimental to a subject, for example, multiplesclerosis, ischemia (e.g. stroke), Alzheimer's disease, Parkinson'sdisease, epilepsy, and spinal cord injury.

BACKGROUND

Multiple sclerosis (MS) is a debilitating disease and is one of theleading causes of non-traumatic neurological disability among the youngpopulation in North America and Europe. The clinical progression of thedisease is variable with ˜85% of patients at diagnosis exhibitingunpredictable and recurring episodes of neurological deficits thatspontaneously subside. Over time, MS patients endure a slow, progressiveand irreversible neurological decline, which may be delayed with earlytherapeutic intervention. The neurodegeneration and ensuing axonal lossin MS patients results in permanent clinical deficits, including limbparalysis, vision loss, spinal cord symptoms, and cognitive deficit.[Noseworthy J H, Lucchinetti C, Rodriguez M, & Weinshenker B G (2000)Multiple sclerosis. The New England Journal of Medicine 343(13):938-952]Because of the long duration of disability and high prevalence amongyoung adults, MS is an enormous public health issue with highsocio-economic burden and significant impact on the quality of life.

The pathophysiology of MS is thought to have an autoimmune origin withmultifocal lesions in the Central Nervous System (CNS) that arecharacterized by inflammation, demyelination, and axonal injury. This iscompounded by the pronounced loss of neuronal connections, which lackthe innate capacity to self-repair and an increased susceptibility ofadult CNS neurons to apoptotic cell death. It is accepted thatirreversible axonal and neuronal loss is a major determinant of theprogressive and permanent neurological impairment in MS patients.[Noseworthy J H, Lucchinetti C, Rodriguez M, & Weinshenker B G (2000)Multiple sclerosis. The New England Journal of Medicine 343(13):938-952]The heterogeneity in the pathology and clinical progression of MS hasled to numerous etiological hypotheses, but none have yet to beconfirmed. Furthermore, it remains unknown whether neurodegenerationprecedes the autoimmune attack in MS or vice versa.

Of interest, elevated circulating iron levels and deposition of iron inthe brains of MS have been reported. [Stankiewicz J M & Brass S D (2009)Role of iron in neurotoxicity: a cause for concern in the elderly?Current Opinion in Clinical Nutrition and Metabolic Care 12(1):22-29;LeVine S M, Bilgen M, & Lynch S G (2013) Iron accumulation in multiplesclerosis: an early pathogenic event. Expert Review of Neurotherapeutics13(3):247-250.] Two genes FPN1 (encodes an iron exporter protein) andHEPC (encodes hepcidin, an enzyme that is crucial in iron regulation)were found to increase the incidence of MS by more than 4-fold and2.5-fold respectively. [Gemmati D, et al. (2012) Polymorphisms in thegenes coding for iron binding and transporting proteins are associatedwith disability, severity, and early progression in multiple sclerosis.BMC Medical Genetics 13:70] Furthermore, reducing levels of ironresulted in reduced disease severity. [Grant S M, Wiesinger J A, Beard JL, & Cantorna M T (2003) Iron-deficient mice fail to develop autoimmuneencephalomyelitis. The Journal of Nutrition 133(8):2635-2638;Stankiewicz J M, Neema M, & Ceccarelli A (2014) Iron and multiplesclerosis. Neurobiology of Aging 35S2:S51-S58; Weigel K J, Lynch S G, &Levine S M (2014) Iron chelation and multiple sclerosis. ASN Neuro6(1):e00136; LeVine S M & Chakrabarty A (2004) The role of iron in thepathogenesis of experimental allergic encephalomyelitis and multiplesclerosis. Annals of the New York Academy of Sciences 1012:252-266]Notably, MS patients undergoing treatment show decreased levels of ironas compared to patients receiving placebo, thus implying a possible rolefor iron in mediating disease progression. [Pawate S, Wang L, Song Y, &Sriram S (2012) Analysis of T2 intensity by magnetic resonance imagingof deep gray matter nuclei in multiple sclerosis patients: effect ofimmunomodulatory therapies. Journal of Neuroimaging: Official Journal ofthe American Society of Neuroimaging 22(2):137-144] The role of iron inMS remains speculative with no clear understanding on whether irondeposition is the result of neurodegeneration or whether it contributesto the development of the disease.

The identification of the MHC II risk allele in MS patients alluded to acentral role played by cluster of differentiation (CD4⁺) T cells in thedevelopment of MS. Analyses of blood and CSF from MS patients furthersuggested that the disease implicated the recruitment of auto-reactiveCD4⁺ T lymphocytes from the periphery to the CNS, where they tethered,rolled, and adhered to endothelial cells lining the blood vessels.[Friese M A, Schattling B, & Fugger L (2014) Mechanisms ofneurodegeneration and axonal dysfunction in multiple sclerosis. NatureReviews. Neurology 10(4):225-238] The subsequent infiltration of thesecells to the parenchyma is associated with breakdown of the blood brainbarrier (BBB). [Gaitan M I, et al. (2011) Evolution of the blood-brainbarrier in newly forming multiple sclerosis lesions. Annals of Neurology70(1):22-29] The initial infiltration of CD4⁺ effector cells of the Thelper 17 (Th17) or Th1 subtypes lead to the secretion ofpro-inflammatory cytokines, such as IL-17α and IFN-γ. [Kebir H, et al.(2007) Human TH17 lymphocytes promote blood-brain barrier disruption andcentral nervous system inflammation. Nature Medicine 13(10):1173-1175]These cytokines are cytotoxic and stimulate the recruitment of otherimmune cells, such as CD8⁺ effector T cells, which are able to furthersecrete cytotoxic cytokines or antigen-presenting cells, such as CD11c⁺cells, which further prime and activate effector T cells within the CNS.This immune cascade together with the activation of CNS residentmicroglia releases cytotoxic cytokines and reactive oxygen or nitrogenspecies to damage the network of supporting oligodendrocytes. Inaddition to this aberrant immune activation, MS patients display adecreased ability to negatively regulate effector T cells, furtherimpacting this immune activation. [Bettelli E, Korn T, & Kuchroo V K(2007) Th17: the third member of the effector T cell trilogy. CurrentOpinion in Immunology 19(6):652-657; Viglietta V, Baecher-Allan C,Weiner H L, & Hafler D A (2004) Loss of functional suppression byCD4+CD25+ regulatory T cells in patients with multiple sclerosis. TheJournal of Experimental Medicine 199(7):971-979] Axonal damage occursearly in demyelinating lesions, which correlate highly with infiltrationof immune cells. [Ferguson B, Matyszak M K, Esiri M M, & Perry V H(1997) Axonal damage in acute multiple sclerosis lesions. Brain: aJournal of Neurology 120 (Pt 3):393-399]

The complexity of MS is highlighted by the heterogeneity of pathologicalpatterns that occur in MS patients. These pathological hallmarks aresubdivided in four distinct patterns. The initial pathological damage ispredominantly regulated by the infiltration of T and B cells at the siteof plaque formation (patterns I and II). Typically these pathologicalpatterns coincide with the novel occurrence of plaques. Subsequently,there is a shift towards decreased cellular infiltration and increasedneurodegeneration represented by sites of hypoxic insult leading toneuronal death and apoptosis (patterns III and IV). MS was originallythought to target white matter tissue, however extensive gray matterlesions have been identified in the early phases of MS progression. Inthe progressive stages of the disease, BBB breakdown does occur, but toa lesser extent in comparison to relapsing-remitting (RRMS).Furthermore, immune cell infiltrates are present in areas that maintainBBB permeability. [Lassmann H, van Horssen J, & Mahad D (2012)Progressive multiple sclerosis: pathology and pathogenesis. NatureReviews. Neurology 8(10:647-656] This suggests that in progressivestages, immune activation takes place within the CNS independently ofperipheral infiltration. At this stage of the disease, patients sufferfrom extensive brain atrophy and dilatation of ventricles.

In MS, loss of BBB integrity occurs early in the disease progression.Breakdown of the BBB primes tissue for the recruitment of leukocytes andsubsequent neuronal damage. While the BBB normally sequesters immunecells outside of the CNS, it can also promote the penetration of immunecells to localized regions of inflammation within the CNS. Early in MS,endothelial cells (ECs) can enhance leukocyte infiltration byup-regulating both E- and P-selectin proteins on their membranes.[Engelhardt B & Ransohoff R M (2005) The ins and outs of T-lymphocytetrafficking to the CNS: anatomical sites and molecular mechanisms.Trends in Immunology 26(9):485-495] Furthermore, ECs can secreteleukocytes attractants such as chemokine ligand 2 (CCL2). [Biernacki K,Prat A, Blain M, & Antel J P (2001) Regulation of Th1 and Th2 lymphocytemigration by human adult brain endothelial cells. Journal ofNeuropathology and Experimental Neurology 60(12):1127-1136] Followingactivation by inflammatory cytokines, ECs can express adhesionmolecules, such as intracellular adhesion molecule-1 (ICAM-1) andvascular adhesion molecule-1, which enhance the extravasation ofleukocytes through ECs. [Alvarez J I, Katayama T, & Prat A (2013) Glialinfluence on the blood brain barrier. Glia 61(12):1939-1958]

In the Experimental Autoimmune Encephalomyelitis (EAE) animal model,which is an accepted animal model of MS, invading immune cells mostprominently target the spinal cord replicating the pathological patternsI and II of MS. [Rangachari M & Kuchroo V K (2013) Using EAE to betterunderstand principles of immune function and autoimmune pathology.Journal of Autoimmunity 45:31-39] The adoptive transfer of CD4⁺ T cellsfrom immunized mice into naïve mice confirmed that EAE was also a CD4⁺ Tcell-mediated disease. [Paterson P Y (1960) Transfer of allergicencephalomyelitis in rats by means of lymph node cells. The Journal ofExperimental Medicine 111:119-136] Initially, IFN-γ producing Th1effector cells were believed to mediate the disease, as adoptivetransfers of Th1 cells induced EAE in mice. However, the discovery thatthe induction of EAE was in fact dependent on a novel cytokine, IL-23,led to the identification of a novel CD4⁺ T cell subtype, the IL-17producing Th17 cells. [Cua D J, et al. (2003) Interleukin-23 rather thaninterleukin-12 is the critical cytokine for autoimmune inflammation ofthe brain. Nature 421(6924):744-748; Becher B, Durell B G, & Noelle R J(2002) Experimental autoimmune encephalitis and inflammation in theabsence of interleukin-12. The Journal of Clinical Investigation110(4):493-497; Park H, et al. (2005) A distinct lineage of CD4 T cellsregulates tissue inflammation by producing interleukin 17. NatureImmunology 6(11):1133-1141] Thus, both Th1 and Th17 CD4⁺ T cell subsetscan mediate EAE. Importantly, both IFN-γ and IL-17 secreting cells wereshown to be enriched in the CSF of MS patients. [Kebir H, et al. (2007)Human TH17 lymphocytes promote blood-brain barrier disruption andcentral nervous system inflammation. Nature Medicine 13(10):1173-1175;Traugott U & Lebon P (1988) Multiple sclerosis: involvement ofinterferons in lesion pathogenesis. Annals of Neurology 24(2):243-251]In EAE, both subtypes of T cells have been shown to promote differentialimmune cell recruitments, with Th1 promoting monocytic inflammation andTh17 promoting neutrophilic infiltrates. [Kroenke M A, Carlson T J,Andjelkovic A V, & Segal B M (2008) IL-12- and IL-23-modulated T cellsinduce distinct types of EAE based on histology, CNS chemokine profile,and response to cytokine inhibition. The Journal of ExperimentalMedicine 205(7):1535-1541] In addition, atypical forms of EAE may arisefrom a shift in the expression of these pro-inflammatory cytokines.Higher expression of IL-17 is associated with more prevalent brainlesions whereas it may be protective in the spinal cord. [Stromnes I M,Cerretti L M, Liggitt D, Harris R A, & Goverman J M (2008) Differentialregulation of central nervous system autoimmunity by T(H)1 and T(H)17cells. Nature Medicine 14(3):337-342] These studies raise thepossibility that T cell subsets may mediate differential functionsdepending on their tissue localization.

In addition to the well-established role of CD4⁺ T-cells in thedevelopment of EAE, studies have also identified other subsets of Tcells that play a crucial role in EAE. Adoptive transfers of CD8⁺ Tcells induce an atypical form of EAE with lesions localized to whitematter of the cerebellum. In addition, this model induces wide-scaleoligodendrocyte death resembling patterns III and IV of MS patients.[Bitsch A, Schuchardt J, Bunkowski S, Kuhlmann T, & Bruck W (2000) Acuteaxonal injury in multiple sclerosis. Correlation with demyelination andinflammation. Brain: a Journal of Neurology 123 (Pt 6):1174-1183] Recentfindings highlight the occurrence of multiple subsets of CD8⁺ T cellspresent in both EAE and MS. Initial studies using CD8^(−/−) knockoutmice have shown increased severity of the disease and more frequentrelapses, indicating a possible regulatory role of CD8⁺ T cells in EAE.[Jiang H, Zhang S I, & Pernis B (1992) Role of CD8+ T cells in murineexperimental allergic encephalomyelitis. Science 256(5060):1213-1215;Koh D R, et al. (1992) Less mortality but more relapses in experimentalallergic encephalomyelitis in CD8−/− mice. Science 256(5060):1210-1213]Furthermore, CD8⁺/CD28^(−/−) T cells have been shown to induceimmunosuppressive phenotypes in EAE by interrupting co-stimulatorymolecule expression on the surface of CD4⁺ T cells. [Montero E, et al.(2004) Regulation of experimental autoimmune encephalomyelitis by CD4+,CD25+ and CD8+ T cells: analysis using depleting antibodies. Journal ofAutoimmunity 23(1):1-7]

Although T cells have been shown to mediate disease, B cells could alsobe involved in the pathophysiology of EAE. MS patients possess higherIgG levels in the CSF compared to age-matched controls, suggesting thepresence of antibody-releasing cells within the CSF. Studies have foundthat B cells enhance EAE severity by promoting differentiation of Th1and Th17 cells. Prior to EAE onset, a regulatory subset of B cells, ableto regulate immune response, exist. This may provide a novel biomarkerfrom which the shift of B cells from regulatory to pathogenic maycorrelate with MS onset. [Fillatreau S, Sweenie C H, McGeachy M J, GrayD, & Anderton S M (2002) B cells regulate autoimmunity by provision ofIL-10. Nature Immunology 3(10):944-950]

SUMMARY

A novel molecular interplay between two members of the RGM family (RGMcand RGMa) is described and its application in modulating thepermeability of the blood brain barrier (BBB), which finds utility inthe treatment or prevention of various diseases and conditions, as wellas in drug delivery applications.

In one embodiment, the disclosure provides use, methods of using and usein the manufacture of a medicament of RGMc, sRGMc or a functionalfragment, variant or mimic thereof for decreasing the permeability ofthe BBB in a subject.

The disclosure further provides, use, methods of use and use in themanufacture of a medicament of RGMc, sRGMc or a functional fragment,variant or mimic thereof for stabilizing or restoring the BBB in asubject.

The disclosure further provides, use, methods of use and use in themanufacture of a medicament of an agent that inhibits RGMa fordecreasing the permeability of the BBB in a subject.

The disclosure further provides, use, methods of use and use in themanufacture of a medicament of an agent that inhibits RGMa forstabilizing or restoring the BBB in a subject.

The disclosure further provides, use, methods of use and use in themanufacture of a medicament of an agent that inhibits RGMc fordisrupting BBB integrity in a subject.

The disclosure further provides, use, methods of use and use in themanufacture of a medicament of an agent, wherein the agent is RGMa,sRGMa, a functional fragment, variant or mimic thereof or stimulatesRGMa, for disrupting BBB integrity in a subject.

The disclosure further provides, use, methods of use and use in themanufacture of a medicament of an agent that inhibits RGMc forincreasing the permeability of the BBB of a subject to a moleculepresent in the blood stream of the subject.

The disclosure further provides, use, methods of use and use in themanufacture of a medicament of an agent, wherein the agent is RGMa,sRGMa, a functional fragment, variant or mimic thereof or stimulatesRGMa, for increasing the permeability of the BBB of a subject to amolecule present in the blood stream of the subject.

Also provided are pharmaceutical compositions comprising RGMc, sRGMc ora functional fragment, variant or mimic thereof and a pharmaceuticallyacceptable carrier or excipient for decreasing the permeability of theBBB in a subject and/or for stabilizing or restoring the BBB in asubject.

Also provided are pharmaceutical compositions comprising an agent thatinhibits RGMa and a pharmaceutically acceptable carrier or excipient fordecreasing the permeability of the BBB in a subject and/or forstabilizing or restoring the BBB in a subject.

Also provided are pharmaceutical compositions comprising an agent thatinhibits RGMc and a pharmaceutically acceptable carrier or excipient fordisrupting BBB integrity in a subject.

Also provided are pharmaceutical compositions comprising an agent,wherein the agent is RGMa, sRGMa, a functional fragment, variant ormimic thereof or stimulates RGMa and a pharmaceutically acceptablecarrier or excipient, for disrupting BBB integrity in a subject.

Also provided are pharmaceutical compositions comprising an agent thatinhibits RGMc and a pharmaceutically acceptable carrier or excipient,for increasing the permeability of the BBB of a subject to a moleculepresent in the blood stream of the subject.

Also provided are pharmaceutical compositions comprising an agent,wherein the agent is RGMa, sRGMa, a functional fragment, variant ormimic thereof or stimulates RGMa and a pharmaceutically acceptablecarrier or excipient for increasing the permeability of the BBB of asubject to a molecule present in the blood stream of the subject.

The disclosure further provides use, methods of using and use in themanufacture of a medicament of RGMc, sRGMc or a functional fragment,variant or mimic thereof for promoting re-myelination in a subject.

The disclosure further provides use, methods of using and use in themanufacture of a medicament of RGMc, sRGMc or a functional fragment,variant or mimic thereof for preventing de-myelination in a subject.

Also provided are pharmaceutical compositions comprising RGMc, sRGMc ora functional fragment, variant or mimic thereof and a pharmaceuticallyacceptable carrier or excipient for promoting re-myelination in asubject.

Also provided are pharmaceutical compositions comprising RGMc, sRGMc ora functional fragment, variant or mimic thereof and a pharmaceuticallyacceptable carrier or excipient for preventing de-myelination in asubject.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A-1B—FIG. 1A shows the structure of RGM proteins. All RGM proteinsshare ˜40% primary amino acid sequence. All members possess anN-terminal signal sequence targeting the proteins to the membrane wherethey are anchored using a GPI-anchor sequence on the most C-terminalportion. All RGMs possess a von Willebrand factor domain whereas bothRGMa and RGMc possess an auto-catalytic RGD motif able to generate asingle-chain and a two-chain membrane-bound protein. RGMb is onlyexpressed as a single-chain membrane-bound protein. Single-chainisoforms can be cleaved from the membrane to generate soluble proteinfragments (asterisks). FIG. 1B shows Neogenin binds RGMa, RGMb and RGMcat various sites. RGMa was shown to bind the Fibronectin type-III₍₃₋₄₎domain of Neogenin, whereas the binding sites for RGMb and RGMc arelocated on the Fibronectin type-III₍₅₋₆₎ domains.

FIG. 2—FIG. 2 shows generation of the experimental autoimmuneencephalomyelitis (EAE) model. As seen on the timeline, an initialinjection of MOG (35-55), in an emulsion with CFA, is injected s.c. inthe hind flank of mice. Subsequently mice are injected with an i.p.injection of pertussis toxin at day 0 and day 2-post-induction of EAE.The typical initial symptoms arise at days 10-12 following induction.This model leads to an acute ascending paralysis (tail>forelimb), whichtypically progresses and eventually plateaus by day 20-22 postinduction.

FIG. 3A-3B—FIG. 3 show RGMc levels in control and EAE animals. (A) RGMcprotein expression in sera of control or EAE mice at days 5, 10, 18, and30 post-induction measured by ELISA; (B) RGMc protein in sera of controland EAE mice at day 18 post-induction analyzed by Western blotting.Quantification of RGMc levels is normalized to transferrin. Data aremeans±SEM (n=3). **p<0.01.

FIG. 4A-4F—FIG. 4 shows sRGMc treatment decreases the clinical severityof EAE-induced mice. Purified sRGMc protein expression was analyzed byWestern blot under non-reducing conditions and by coomassie staining(A). Body condition score in EAE-induced animals treated with vehicle orinjected with 20 mM sRGMc intravenously (i.v.) every 3 days for theduration of disease course (arrowheads) (B) or 20 mM sRGMcintraperitoneally (i.p.) injected at days 3, 6, and 9 for the durationof disease course (arrowheads) (C). Relative incidence of EAE in animalstreated with vehicle or 20 mM sRGMc by i.p. (D). Day of onset of EAE inanimals treated with vehicle or 20 mM sRGMc i.p. (E). sRGMc treatment isstable for 24 h post treatment and restores sRGMC levels to controllevels, as observed by ELISA (F). Data are means±SEM (n=3-15 animals pergroup). **p<0.01; ***p<0.0001.

FIG. 5A-5C—FIG. 5 shows RGMc knockout animals (KO) could be susceptibleto EAE. (A) Body condition score in EAE-induced wild type (WT) animalsor selective homozygous deletion of RGMc (129S-Hfe2^(tm1Nca)/J) (RGMcKO). (B) Day of onset of EAE in RGMc KO and control (WT). (C) EAEincidence in RGMc knockout animals (KO) or control (WT). Data aremeans±SEM (n=10 animals per group).

FIG. 6A-6D—FIG. 6 shows sRGMa is up-regulated in EAE and interferes withsRGMc for binding to Neogenin. (A) sRGMa expression in the sera ofcontrol mice and in mice induced with EAE at days 5, 10, 18, and 30analyzed using an RGMa ELISA kit as described in the Examples. (B) Bodycondition scores of 6-week old EAE-induced mice treated with PBS(Vehicle), every three days with 20 mM sRGMc, or with 20 mM sRGMc inconjunction with 40 mM sRGMa. (C) Binding of Neogenin to bovine serumalbumin (Neogenin+BSA), to RGMa (Neogenin+RGMa), and to RGMc(Neogenin+RGMc) in RGMa-AP and in RGMc-AP. Constructs overexpressingHis-tagged sRGMa, sRGMc and the extracellular domains of Neogenin(Ec-Neogenin) were coated onto Poly-L-Lysine coated wells. AlkalinePhosphatase (AP)-tagged proteins (EC-Neogenin or RGMa or RGMc) were thenadded to each well and incubated at 37° C. for 1 h. Each well was washedand equilibrated with AP developing buffer (100 mM NaHCO₃, 1 mM MgCl₂).The reaction was initiated using AP developing buffer supplemented withp-nitrophenyl phosphate and then stopped following color development byadding 50 μl (0.1M) NaOH. The absorbance of each reaction was measuredusing a microplate autoreader at 405 nm as described in the Examples.(D) Binding of Neogenin-AP to bovine serum albumin (Neogenin-AP+BSA), toRGMa (Neogenin-AP+RGMa), and to RGMc (Neogenin-AP+RGMc). Competitionassay was performed as described in the Examples and above but in whichthe constructs were reversed. Data are means±SEM (n=3-10 animals pergroup, A-B); n=3 in triplicates (binding assays, C-D). *p<0.05,**p<0.01.

FIG. 7—FIG. 7 shows sRGMc treatment has no effect on naïve immune cellpopulations. Mice induced with EAE were sacrificed at 10 dayspost-induction and splenocytes were harvested and stained as describedin the Examples. No difference was observed in the percentage of Bcells, T cells, or MHC II on B cells in RGMc-treated versusvehicle-treated animals (n=15)

FIG. 8A-8D—FIG. 8 shows sRGMc treatment has no effect on naïveantigen-presenting cells. Mice induced with EAE were treated with PBS(Vehicle) or with 20 mM sRGMc (RgMc) and were sacrificed 10 dayspost-induction. Splenocytes were harvested and analyzed as described inthe Examples for the percentage of (A) CD11c (dendritic cell), (B) CD11b(myeloid cell), and (C) coactivation markers CD80 on B cells and (D)coactivation markers CD86 cells on B cells. Data are means±SEM (n=15animals per group).

FIG. 9A-9C—FIG. 9 shows sRGMc treatment has no effect on the adhesionproperties of naïve T and B cells. Mice induced with EAE were treatedwith PBS (Vehicle) or with 20 mM sRGMc (RgMc) and were sacrificed 10days post-induction. Splenocytes were harvested and analyzed asdescribed in the Examples for (A) ICAM-1 expression on B cell, (B)ICAM-1 expression on CD8⁺ T cells, (C) ICAM-1 and CD62L expressions inCD4⁺T cells. Data are means±SEM (n=15 animals per group).

FIG. 10A-10B—FIG. 10 shows sRGMc treatment has no effect on activatedimmune cells. Mice induced with EAE were treated with either PBS(Vehicle) or with sRGMc (RgMc) and sacrificed 10 days post induction.Splenocytes or draining lymph nodes (caudal, sciatic, lumbar) cells wereharvested, stimulated with PMA and ionomycin as described in theExamples. Expression of activated CD4⁺T cells secreting IL-17A or IFN-γin (A) splenocytes or (B) Draining lymph nodes (caudal, sciatic, lumbar)cells. Data are means±SEM (n=15 animals per group).

FIG. 11A-11B—FIG. 11 shows sRGMc treatment has no effect onantigen-specific immune cells. Mice induced with EAE were treated withPBS or RGMc. Splenocytes were harvested 10 days post-induction asdescribed in the Examples. (A) CD4⁺T cell proliferation as measuredusing CFSE incorporation in splenocytes treated with 0 or 30 μg/mLMOG(35-55) (B) Levels of IL-17 and IFN-γ in splenocytes pulsed with 0,30, or 100 μg/mL MOG(35-55). Data are means±SEM (n=15 animals pergroup).

FIG. 12—FIG. 12 shows sRGMc treatment does not alter the cytokinesecretion of MOG-pulsed splenocytes. Concentrations of IL17, IFN-γ,TNFα, and IL6 measured in splenocytes harvested from EAE-induced micetreated with either PBS (Vehicle) or RGMc and pulsed with MOG for 96 h.Cytokine secretion was analyzed using an ELISA as described in theExamples. Data are means±SEM (n=15 animals per group).

FIG. 13A-13C—FIG. 13 shows sRGMc treatment reduces the number ofcellular infiltrates and the extent of de-myelination in EAE-inducedmice. Cervical cord sections were isolated from mice induced with EAE(day 18) and treated with either PBS (Vehicle) or sRGMc (RGMc) andstained with H&E in combination with Luxol Fast Blue as described in theExamples. (A) Representative images of cervical spinal cord sectionsshowing less demyelinating foci in EAE-induced mice treated with sRGMc.(B) Quantification of Luxol fast blue staining (mean pixel intensity) invehicle-treated (normalized to sham animals) and in RGM-treated EAEanimals. (C) Inflammatory index scores in cervical sections invehicle-treated and in RGM-treated EAE animals. Data are means±SEM (n=6animals per group). **p<0.01.

FIG. 14A-14B—FIG. 14 shows sRGMc reduces the amount of immuneinfiltrates in the spinal cord of EAE-induced mice. Cervical cordsections were isolated from mice induced with EAE (day 18) and treatedwith either PBS (Vehicle) or sRGMc (RGMc), cryo-sectioned and stainedwith CD3, CD11b, or B220 surface markers as described in the Examples.Representative images (A) and quantification of cells in 7 regions ofspinal cord sections (B) stained with CD3, CD11b, or B220 surfacemarkers. Data are means±SEM (n=6 animals per group). ***p<0.001.

FIG. 15A-15D—FIG. 15 shows sRGMc decreases blood brain barrier (BBB)endothelial cell permeability by interfering with RGMa-mediated Rhoactivation. (A) Neogenin expression was analyzed on the membrane ofb.end3 cells using immunohistochemical staining and by Western blot. (B)Crystal violet staining showing the b.end3 cell monolayer, (C)Representative diagram of the boyden chamber set up and (D) BBBpermeability in b.end3 cells. Serum-starved cells were incubated for 4 hwith RGMa (10 μg/ml), C3 Transferase (10 μg/mL) or Y27632 (10 μM) orsRGMc (10 μg/mL) and were then incubated with sRGMa (5 μg/mL) for 18 hfollowed by seeding of Dextran-FITC onto the upper chamber.Extravasation was measured over a period of 60 min as described in theExamples. n=3, performed in triplicates.

FIG. 16A-16B—FIG. 16 shows Neogenin expression in endothelial cells inmouse spinal cord. (A) Composition images of cervical spinal cordsections of C57/BL6 mice stained for Neogenin shown in red (color notshown), for endothelial cell stained with anti-CD31 antibody shown ingreen (color not shown) and for nucleus stained with DAPI in blue (colornot shown) (B) Merged image of Neogenin staining, CD31 staining and DAPIstaining, indicated by alteration in color (color not shown), whichindicated colocalization of Neogenin and CD31 (endothelial cell marker).

FIG. 17A-17B—FIG. 17 shows sRGMc reduces BBB permeability. (A)Immunohistochemical staining of cervical spinal cords of 18 daysEAE-induced mice treated every 3 days with sRGMc or Vehicle. Shown arerepresentative images of fibrinogen extravasation in red (color notshown) and arrowheads around blood vessels stained with anti-CD31antibody in green (color not shown) and asterisks, nuclei stained withDAPI in blue (color not shown), and merged images of fibrinogen stainingand CD31 staining indicated by alteration in color (color not shown),which indicated colocalization of fibrinogen and blood vessels. Scalebars, 50 μm. (B) Quantitative analysis of perivascular extravasation offibrinogen by measuring the relative pixel intensity in sRGMc-treatedand vehicle-treated animals (n=6).

FIG. 18A-18B—FIG. 18 shows RGMc protects BBB integrity. Mice weretreated with RGMa (70 ug), RGMa (70 ug)+RGMc (70 ug), or Vehicle (1%BSA) 18 h prior to widefield imaging (100-300 ms exposure) prior to andfollowing intravenous injection of Texas Red dye (0.5 mg) over time. (A)Representative images showing the leakage of Texas Red dye. (B)Quantification of the leakage of Texas Red dye (measured as the numberof leakage sites/mm²).

FIG. 19—FIG. 19 shows a representative immunoblot of Claudin 5expression in endothelial cells treated with PBS, RGMc, RGMa orRGMc+RGMa. End3-endothelial cells (ECs) were used to generate an invitro model of the human endothelial cell barrier lining the choroidplexus. b.end3-ECs were plated at 2×10⁵ cells per dish in DMEM, and wereallowed to reach confluency at which point they were serum-starved for 6h. Cells were pretreated for 6 h with PBS, RGMc, RGMa or RGMc+RGMa.Western blotting was performed on the lysates with Claudin-5 and GAPDHantibodies. This shows that RGMa induces a downregulation of Claudin 5,which is restored by RGMc.

FIG. 20—FIG. 20 shows CD4 positive transplanted cells are observed incells treated with Vehicle but not RGMc. EAE was passively induced in6-8 week old C57Bl/6 mice and sacrificed. Donor animals were immunizedwith 100 ug MOG(35-55) and sacrificed at day 10. Splenocytes wereharvested and re-stimulated for three days with 33 ug/mL MOG(35-55) inthe presence of 20 ng/ml rmIL-23. Cells were then labelled with CellVueMaroon and injected into recipient animals treated with RGMc or vehicleas well as PTx on day 0 and day 2. Recipient animals were treated every3 days (with PBS or RGMc) and sacrificed on day 8 and spinal cordssectioned for immunofluorescence staining with CD4 antibody, or usingthe labelling from cell vue. This shows that in PBS, but not in RGMc,CD4 positive transplanted cells were penetrating the BBB.

FIG. 21A-21C—FIG. 21 shows the effects of RGMc on BBB integrity in vivo.BBB integrity was assessed in mice treated with PBS (Vehicle), RGMa,RGMc, RGMa+ immunoglobulin domain of Neogenin (4Ig). Widefield imagingwas then carried out under a Olympus microscope (BX6 1WI) prior to andfollowing the injection of Texas red dye (100 uL, I.V., 5 mg/ml) using100-300 ms exposure. Recording was performed from 0 min to 35 minfollowing injection. Texas dye leakage was also assessed in RGMcknockout (KO) and wild type (WT) mice. The leakage of Texas-red dye wasquantified by a blind experimenter measuring the number of leakagesites/mm². (A) Representative images of Texas red dye: PBS (Vehicle),RGMa, RGMa+RGMc and RGMa+4Ig (immunoglobulin domain of Neogenin).Quantification of the leakage of Texas Red dye in animals treated with(B) PBS (Vehicle), RGMa or RGMa+RGMc or in RGMc KO and WT mice or (C)PBS (Vehicle), RGMa, RGMa+RGMc or RGMa+4Ig.

FIG. 22—FIG. 22 shows sRGMc treatment promotes extensive remyelinationin cerebral explants. Explants were demyelinated with lysolecithin andtreated with sRGMc. Explants were stained for myelin basic protein(MBP).

FIG. 23—FIG. 23 shows treatment with sRGMc promotes regeneration in anoptic crush model. Optic nerves were treated with sRGMc and stained foraxonal fibers with GAP43. Line represents the site of injury.

FIG. 24A-24B—FIG. 24 shows treatment with sRGMc reduces Evans Bluepenetration into CNS tissue in a middle cerebral artery occlusion model.

FIG. 25—FIG. 25 shows treatment with sRGMc promotes functional recoveryafter middle cerebral artery occlusion.

DETAILED DESCRIPTION

The RGMa/Neogenin pathway is involved in neuronal survival, as well asregenerative failure and the immune response in EAE animals. Two othermembers of the RGM family have also been characterized in mammals: RGMb(DRAGON), and RGMc (Hemojuvelin, HFE2). These proteins share ˜40%identity in primary amino acid sequences and all three express anN-terminal signal peptide sequence followed by anarginine-aspargine-aspartic acid (RGD) motif. Other key structuresinclude the presence of a partial von Willebrand type D domain and acarboxy-terminal GPI anchor [Severyn C J, Shinde U, & Rotwein P (2009)Molecular biology, genetics and biochemistry of the repulsive guidancemolecule family. The Biochemical Journal 422(3):393-403] (FIG. 1A). BothRGMa and RGMc can undergo autocatalytic cleavage generating amembrane-bound isoform that can further undergo post-translationalcleavage to secrete soluble fragments. [Tassew N G, Charish J, Seidah NG, & Monnier P P (2012) SKI-1 and Furin generate multiple RGMa fragmentsthat regulate axonal growth. Developmental Cell 22(2):391-402; Nili M,David L, Elferich J, Shinde U, & Rotwein P (2013) Proteomic analysis andmolecular modelling characterize the iron-regulatory proteinhaemojuvelin/repulsive guidance molecule c. The Biochemical Journal452(1):87-95] These proteins regulate complex biological activities,including cell adhesion, axonal outgrowth, axonal guidance, immuneregulation, and systemic iron regulation. All of these functions aremediated through the trans-membrane receptor Neogenin, which is broadlyexpressed in the body.

Neogenin is a type-I transmembrane protein that is homologous to thewell-known tumor suppressor receptor, Deleted in Colorectal Cancer(DCC). Both of these receptors possess an extracellular domaincontaining four immunoglobulin (4Ig) domains and six-fibronectin typeIII (FNIII) domains (FIG. 1B). The intracellular regions differ betweenthe two receptors except for 3 conserved regions termed P1, P2, and P3.Unlike DCC, Neogenin is expressed broadly outside of the CNS in tissuesincluding: lung, heart, gut, kidney, liver, skeletal muscle, and bone.The well-known Netrin-1 ligand, known to bind DCC at the FNIII₍₄₋₅₎domains was also shown to bind to Neogenin at the same location [Cole SJ, Bradford D, & Cooper H M (2007) Neogenin: A multi-functional receptorregulating diverse developmental processes. The International Journal ofBiochemistry & Cell Biology 39(9):1569-1575] and promote attraction ofsupraoptic axons in the Xenopus forebrain. [Xu K, et al. (2014) Neuralmigration. Structures of netrin-1 bound to two receptors provide insightinto its axon guidance mechanism. Science 344(6189):1275-1279] Neogeninwas also shown to bind RGM proteins, with the RGMa-Neogenin interactionfirst identified through its chemo-repulsive effect in temporal retinalaxons of the chick anterior optic tectum. [Wilson N H & Key B (2006)Neogenin interacts with RGMa and netrin-1 to guide axons within theembryonic vertebrate forebrain. Developmental Biology 296(2):485-498] Asshown in FIG. 1B, the binding site of RGMa on Neogenin resides withinthe FNIII₍₃₋₄₎ domain whereas the binding sites for RGMb and RGMc arelocated on the FNIII₍₅₋₆₎ domains. [Tassew N G, Charish J, Seidah N G, &Monnier P P (2012) SKI-1 and Furin generate multiple RGMa fragments thatregulate axonal growth. Developmental Cell 22(2):391-402 Yang F, West AP, Jr., Allendorph G P, Choe S, & Bjorkman P J (2008) Neogenin interactswith hemojuvelin through its two membrane-proximal fibronectin type IIIdomains. Biochemistry 47(14):4237-4245; Bell C H, et al. (2013)Structure of the repulsive guidance molecule (RGM)-neogenin signalinghub. Science 341(6140:77-80] Furthermore, the functions of both RGMb andRGMc are mediated through Neogenin signaling. [Enns C A, Ahmed R, &Zhang A S (2012) Neogenin interacts with matriptase-2 to facilitatehemojuvelin cleavage. The Journal of Biological Chemistry287(42):35104-35117; Ma C H, et al. (2011) The BMP coreceptor RGMbpromotes while the endogenous BMP antagonist noggin reduces neuriteoutgrowth and peripheral nerve regeneration by modulating BMP signaling.The Journal of Neuroscience: the Official Journal of the Society forNeuroscience 31(50):18391-18400]

The discovery of RGMc, also known as Hemojuvelin, was completed bymapping the gene locus associated with an autosomal recessive disease,juvenile hemochromatosis (JH). [Enns C A, Ahmed R, & Zhang A S (2012)Neogenin interacts with matriptase-2 to facilitate hemojuvelin cleavage.The Journal of Biological Chemistry 287(42):35104-35117] JH presents asan early onset of iron overload typically in the first and second decadeof life, and is caused by the lack of function of a liver-derivedenzyme, hepcidin. [Ganz T (2007) Molecular control of iron transport.Journal of the American Society of Nephrology: JASN 18(2):394-400] RGMcknockout mice develop iron overload and a decreased hepcidin expression,which is similar to observations in JH. [Core A B, Canali S, & Babitt JL (2014) Hemojuvelin and bone morphogenetic protein (BMP) signaling iniron homeostasis. Frontiers in Pharmacology 5:104] RGMc protein alsoundergoes complex post-translational processing. The presence of anautocatalytic sequence results in two cell membrane proteins: i) asingle-chain 50 KDa; and 2) a two-chain 30 KDa protein linked to a 20KDa protein by di-sulfide bonds. The single-chain protein can be furtherprocessed by a pro-protein convertase, Furin, to release a 50 KDafragment. [Kuninger D, Kuns-Hashimoto R, Nili M, & Rotwein P (2008)Pro-protein convertases control the maturation and processing of theiron-regulatory protein, RGMc/hemojuvelin. BMC biochemistry 9:9]Furthermore, single-chain RGMc can be cleaved by a serine-proteaseMatriptase-2 (MT2) to release a 36 KDa fragment. [Enns C A, Ahmed R, &Zhang A S (2012) Neogenin interacts with matriptase-2 to facilitatehemojuvelin cleavage. The Journal of Biological Chemistry287(42):35104-35117] In hepatocytes, single-chain membrane-bound RGMcacts as a co-receptor for BMP-6 and induces hepcidin expression.[Andriopoulos B, Jr., et al. (2009) BMP6 is a key endogenous regulatorof hepcidin expression and iron metabolism. Nature Genetics41(4):482-487; Meynard D, et al. (2009) Lack of the bone morphogeneticprotein BMP6 induces massive iron overload. Nature Genetics41(4):478-481] Interestingly, the double-, but not the single-chain RGMcbinds to Neogenin. [Yang F, West A P, Jr., Allendorph G P, Choe S, &Bjorkman P J (2008) Neogenin interacts with hemojuvelin through its twomembrane-proximal fibronectin type III domains. Biochemistry47(14):4237-4245; Kuns-Hashimoto R, Kuninger D, Nili M, & Rotwein P(2008) Selective binding of RGMc/hemojuvelin, a key protein in systemiciron metabolism, to BMP-2 and neogenin. American Journal of Physiology.Cell Physiology 294(4):C994-C1003] However, Neogenin knockout animalsexperience iron overload, have low levels of hepcidin, and reduced BMPsignaling. [Lee D H, et al. (2010) Neogenin inhibits HJV secretion andregulates BMP-induced hepcidin expression and iron homeostasis. Blood115(15):3136-3145] The mechanism by which Neogenin regulates iron ispoorly understood with reports that it increases [Enns C A, Ahmed R, &Zhang A S (2012) Neogenin interacts with matriptase-2 to facilitatehemojuvelin cleavage. The Journal of Biological Chemistry287(42):35104-35117] and decreases [Lee D H, et al. (2010) Neogenininhibits HJV secretion and regulates BMP-induced hepcidin expression andiron homeostasis. Blood 115(15):3136-3145] RGMc cleavage at themembrane. Soluble fragments of RGMc (sRGMc) play crucial roles in ironregulation. Whereas membrane-bound RGMc promotes hepcidin expression,sRGMc inhibits hepcidin expression by competing with BMP receptors forBMP ligands. [Babitt J L, et al. (2007) Modulation of bone morphogeneticprotein signaling in vivo regulates systemic iron balance. The Journalof Clinical Investigation 117(7):1933-1939]

RGMa serves a pivotal role in the proper development of the CNS, but itsrole ex-CNS has been largely understudied. Reports of increased RGMaexpression on activated macrophages following spinal cord injury raiseda potential route by which RGMa may mediate its chemo-repulsive signalon regenerating axonal fibers following injury. [Hata K, et al. (2006)RGMa inhibition promotes axonal growth and recovery after spinal cordinjury. The Journal of Cell Biology 173(1):47-58; Kitayama M, Ueno M,Itakura T, & Yamashita T (2011) Activated microglia inhibit axonalgrowth through RGMa. PloS One 6(9):e25234] Indeed, in vitro co-culturesof mouse cortical neurons with macrophages supplemented with LPSinhibited neurite outgrowth and growth cone collapse. This RGMa effectis mediated through its interaction with Neogenin since knockdown ofNeogenin decreased the chemorepulsive effect of activated macrophages.[Kitayama M, Ueno M, Itakura T, & Yamashita T (2011) Activated microgliainhibit axonal growth through RGMa. PloS One 6(9):e25234] Thus, RGMa isexpressed on the surface of macrophages at the site of injury and isable to repulse regenerating fibers.

RGMa was recently linked to murine EAE, shifting our understanding ofthe physiological role of RGMa. This study was extended by genotypingpolymorphisms in the RGMa locus of MS patients, which revealed to behighly correlative and followed a female bias, as observed in clinicalonsets of the disease. RGMa polymorphisms correlated with elevated TNF-αand IFN-γ in the CSF and the peripheral blood mononuclear cells (PBMCs)of MS patients. [Nohra R, et al. (2010) RGMA and IL21R show associationwith experimental inflammation and multiple sclerosis. Genes andImmunity 11(4):279-293] In addition, soluble fragments of RGMa werefound in the cerebrospinal fluid of RRMS patients, which decreased inpatients undergoing intrathecal corticosteroid triamcinolone acetonidetreatment. [Demicheva E, et al. (2015) Targeting Repulsive GuidanceMolecule A to Promote Regeneration and Neuroprotection in MultipleSclerosis. Cell Reports] LPS administration upregulated full-length andauto-catalytically cleaved forms of RGMa on the surface of bonemarrow-derived dendritic cells, whereas its receptor, Neogenin, wasexpressed on the surface of CD4⁺ T cells. Western blot analysisidentified a small GTPase, Rap1, which upon RGMa expression becomeselevated leading to an increase in T cell adhesion (observed viaincreased ICAM-1 expression). [Muramatsu, R., Kubo, T., Mori, M.,Nakamura, Y., Fujita, Y., Akutsu, T., Okuno, T., Taniguchi, J.,Kumanogoh, A., Yoshida, M., et al. (2011). RGMa modulates T cellresponses and is involved in autoimmune encephalomyelitis. Nat. Med. 17,488-494] This finding highlights a possible role for the RGMa-Neogeninsignaling pathway in immune cell priming and activation.

RGMa has been further implicated in immune regulation through itsability to transiently (for 8 hours) hinder leukocyte extravasation inan in vivo model of Zymosan-A-induced peritonitis. RGMa is expressed incytokeratin-positive epithelial cells in addition to polymorphonuclearleukocytes that expressed both RGMa and Neogenin. [Mirakaj V, et al.(2011) Repulsive guidance molecule-A (RGM-A) inhibits leukocytemigration and mitigates inflammation. Proceedings of the NationalAcademy of Sciences of the United States of America 108(16):6555-6560]Moreover, Neogenin knockout animals lost the ability to develop immuneresponse following an acute peritonitis model further suggest that theRGMa/Neogenin signaling pathway plays a role in acute inflammation.[Konig K, et al. (2012) The axonal guidance receptor neogenin promotesacute inflammation. PloS one 7(3):e32145] Taken together, the expressionof RGMa on peripheral immune cells can modulate immune activationthrough dendritic cell-T cell signaling, or by inhibiting theinfiltration of immune cells through epithelial cells. The potential ofRGMa signaling in MS goes far beyond immune modulation. Inhibition ofRGMa signaling promotes axonal regeneration in several spinal cordinjury models as well as optic nerve injury models. [Hata K, et al.(2006) RGMa inhibition promotes axonal growth and recovery after spinalcord injury. The Journal of Cell Biology 173(0:47-58] Despite the recentfocus on RGMa and its role as an immune modulator, the mechanism bywhich RGMa mediates its actions on the immune system is still poorlyunderstood.

Thus, while RGMa and its interaction with Neogenin have been shown toplay a crucial role in immune activation and priming, yet these studiesfail to identify the role of soluble RGMa in the development of MS.Likewise, RGMc is also processed and co-exists with soluble RGMa in theserum. Both soluble proteins were shown to mediate their signal throughtheir interactions with Neogenin.

The examples below evidence that the interplay between levels of RGMaand RGMc is crucial for the development of autoimmune diseases, such asMS. The role of RGMc in the development of autoimmune diseases wasexamined in the context of a mouse animal model of MS, i.e., EAE.Specifically: (1) if RGMc and RGMa levels are altered in the sera ofEAE-induced mice, (2) the relationship between RGMa and RGMc in thedevelopment of EAE, (3) if RGMc modulates the molecular activation ofthe adaptive immune system in the development of EAE, and (4) if RGMcand/or RGMa modify the blood brain barrier (BBB) was examined.

As detailed in the Examples, the present inventors show that sRGMapresent in the serum is up regulated following the induction of EAE andis able to increase the blood-brain EC barrier permeability, therebyincreasing the infiltration of leukocytes within the CNS. The thirdmember of the RGM family, RGMc, undergoes post-translational processingto generate soluble fragments present in the serum. As detailed in theExamples, a previously uncharacterized function for the iron regulatoryprotein RGMc is identified, whereby its expression in the sera ofEAE-induced mice is significantly down regulated. Over-expression ofsRGMc reduced the clinical severity in EAE-induced mice. RGMc knockoutanimals develop a worse disease progression that their wild-typecontrol. Moreover, sRGMc treatment both diminished the extent ofblood-borne proteins and the amount of leukocytes extravasating in theCNS. Lastly, it is shown in the Examples that this effect is mediatedthrough the competition of these proteins for their receptor Neogenin onthe endothelium of the BBB. Together, this data evidences a novelmolecular interplay between two members of the RGM family and theirregulation in the development of EAE.

As shown in the Examples, following the induction of EAE, both RGMa andRGMc levels are modulated prior to the onset of symptoms. In particular,sRGMc levels are significantly reduced in the sera of EAE mice. Thus,sRGMc levels are associated with disease progression, as exogenousexpression of sRGMc in these EAE mice reverts the clinical severity.

That a complex physiological interplay exists between sRGMa and sRGMc issupported by the observation that sRGMa co-treatment completelyabolishes EAE paralysis in sRGMc-treated mice. Using an in vitrocompetition assay a novel molecular interplay between these moleculeswas identified whereby both proteins compete for the same receptor,Neogenin. The binding site of RGMa (FNIII₍₃₋₄₎) differs from that ofRGMc on Neogenin (FNIII₍₅₋₆₎). Thus, without being bound by a theory, itis believed that these molecules must regulate each other's bindingthrough either negative allosteric modulation or by steric hindrance onNeogenin.

The data provided in the Examples evidences that sRGMc reduces BBBpermeability as shown by a decrease in endogenous fibrinogenextravasation in EAE mice. Without being bound by a theory, it isproposed that sRGMa increases BBB permeability by activating the smallGTPase RhoA. This small GTPase family comprising of RhoA, Rac1, andCDC42 links membrane receptors to cytoskeletal remodeling via actinassembly and disassembly. Indeed, the repulsive function of RGMa onprojecting growth cones is mediated by the downstream activation ofRhoA. [Hata K, et al. (2006) RGMa inhibition promotes axonal growth andrecovery after spinal cord injury. The Journal of Cell Biology173(1):47-58] In addition, numerous axon guidance molecules have beenshown to activate the Rho signaling pathway to mediate their guidancecues. [Mueller B K, Mack H, & Teusch N (2005) Rho kinase, a promisingdrug target for neurological disorders. Nature Reviews. Drug Discovery4(5):387-398] The direct downstream effector of RhoA is Rho kinase(ROCK), which is a serine/threonine protein kinase.

The work presented in the Examples is the first to identify the role ofRGMc in the development of EAE. An observation that soluble RGMfragments possess contrasting expression patterns during the course ofEAE led to an investigation of the role of exogenous sRGMc in thedevelopment of EAE. sRGMc-treated animals developed a delayed onset, adecreased percentage of incidence, and a diminished clinical severity ofthe disease course. Moreover, RGMc knockout animals developed a moresevere disease progression than wild-type animals. As detailed in theExamples, an in vitro competition assay uncovered a molecular mechanismwhereby sRGMc was shown to compete with sRGMa for the binding toNeogenin. This observation was confirmed in vivo by treatingRGMC-treated animals in conjunction with sRGMa and observing a worseningdisease phenotype. These findings prompted investigation of a role forRGMc in the infiltration of leukocytes within the CNS. Further, it wasdemonstrated that RGMc strengthened the integrity of the BBB in EAE.

Based on the published literature, the role of RGMc was exclusivelybelieved to regulate iron expression. The Examples below provideevidence for the role of this protein in mediating RGMa signaling. Thefinding that sRGMc is able to protect and stabilize the BBB provideswider implications than MS as several disorders exhibit a breach of theBBB.

The blood-brain barrier (BBB) is a highly selective permeability barrierthat separates the circulating blood from the brain extracellular fluidin the CNS. The BBB, which is formed by brain endothelial cells, allowsthe passage of water, some gases, and lipid-soluble molecules by passivediffusion, as well as the selective transport of molecules such asglucose and amino acids that are crucial to neural function, whilerestricting the diffusion of microscopic objects (e.g., bacteria orcells such as leukocytes) and large or hydrophilic molecules into thecerebrospinal fluid (CSF). Most pharmaceutical agents cannot passthrough the BBB presenting delivery challenges.

As used herein “increasing the permeability of the BBB” refers toincreasing the amount or size of molecules or microscopic objectstransported across the blood brain barrier, while conversely “decreasingthe permeability of the blood brain barrier” refers to decreasing theamount or size of molecules or microscopic objects transported acrossthe BBB.

Stabilizing or restoring the BBB in a subject refers to stopping orretarding the rate of increases (stabilizing) or reversing increases(restoring) in BBB permeability.

As used herein, “polypeptide” and “protein” are used interchangeably andmean proteins, protein fragments, modified proteins, amino acidsequences and synthetic amino acid sequences. As used herein, a“variant” describes a peptide or polypeptide that differs from areferenced peptide or polypeptide in amino acid sequence by insertion,deletion, or conservative substitution of amino acids, or inpost-translational processing, but that retains the relevant biologicalactivity of the referenced peptide or polypeptide.

In certain embodiments, the method may modulate the interaction betweenRGMa and Neogenin. The agent may be an antibody, a peptide agent, acholesterol-lowering agent, or any combination thereof. The peptideagent may be an RGMa peptide, a Neogenin peptide, a Noggin peptide,fragment thereof, variant thereof, or any combination thereof.

The Neogenin peptide may include fragments of Neogenin, variants ofNeogenin, or any combination thereof. The Neogenin peptide may includetwo immunoglobulin-like domains of Neogenin, fragments thereof, variantsthereof, or any combination thereof. The Neogenin peptide may includethree immunoglobulin-like domains of Neogenin, fragments thereof,variants thereof, or any combination thereof. The Neogenin peptide mayinclude four immunoglobulin-like domains of Neogenin, fragments thereof,variants thereof, or any combination thereof. Four immunoglobulin-likedomains of Neogenin may also be referred to herein as the 4Ig domain ofNeogenin. Accordingly, the Neogenin peptide may include the 4Ig domain,fragment thereof, variant thereof, or any combination thereof.

The RGMa peptide may include fragments of RGMa, variants of RGMa, or anycombination thereof. The RGMa peptide may be any fragment of RGMa thatinteracts with the 4Ig domain of Neogenin. The RGMa peptide may containany secondary structure that may be required for the cis interaction tooccur between RGMa and Neogenin.

The RGMa peptide sequence is publicly available and accessible athttp://www.uniprot.org/uniprot/Q96B86 for H. sapiens andhttp://www.uniprot.org/uniprot/Q6PCX7 for M. musculus. In certainembodiments, a functional fragment of RGMa may be used, which cancomprise a fragment of RGMa at least 50 amino acids in length thatinteracts with Neogenin. sRGMa can be prepared according to methodsdescribed in Tassew N G, Charish J, Seidah N G, Monnier P P. SKI-1 andFurin generate multiple RGMa fragments that regulate axonal growth. DevCell. 2012 Feb. 14; 22(2):391-402. doi: 10.1016/j.devce1.2011.11.022.PubMed PMID: 22340500.

Peptides that act similar to the RGMa peptide, i.e., blocking ordisrupting the interaction between RGMa and Neogenin, may include apeptide from RGMc.

The RGMc peptide sequence is publicly available and is accessible usingUniprot AC/ID identifier Q6ZVN8 on the Uniprot website for H. sapiensand using Uniprot AC/ID identifier Q7TQ32 on the Uniprot website for M.musculus. In certain embodiments, a functional fragment of RGMc may beused, which can comprise a fragment of RGMc at least 50 amino acids inlength that interacts with Neogenin.

The method may modulate the interaction between RGMa and Neogenin byadministering the Noggin peptide to the subject. The Noggin peptide maydisrupt or block the cis interaction.

The Noggin peptide may include fragments of Noggin, variants of Noggin,or any combination thereof. The Noggin peptide may include the aminoacid sequence of Accession No. AAA83259.

The cholesterol-lowering agent may be, but is not limited to,methyl{circumflex over ( )}-cyclodextrin (MpCD), cholesterol oxidase(CO), AY-9944, a statin, a subtisilin/kexin type 9 (PCK9) inhibitor,nystatin, filipin, proprotein convertase, BM 15.766, alkylphospholipidanalogs (e.g., miltefosine, edelfosine, and perifosine), or anycombination thereof.

The antibody may be directed against the RGMa peptide described above.This RGMa peptide contains any secondary structure that may be requiredfor the cis interaction to occur between RGMa and Neogenin. Accordingly,the antibody directed against the RGMa peptide may specificallyrecognize and selectively bind this secondary structure.

The antibody may specifically recognize and selectively bind the 4Igdomain of Neogenin. The antibody may specifically recognize andselectively bind amino acids 1 to 383 of Accession No. AAC59662 or aminoacids 1 to 417 of Accession No. AAI43272.

In certain embodiments, methods may modulate the interaction betweenRGMc and Neogenin. Agents for use in such methods can include RGMa,sRGMa, a functional fragment, variant or mimic thereof or an agent thatstimulates RGMa production or enhances RGMa binding to Neogenin.

In other embodiments, methods as disclosed herein may be effectedthrough gene therapy, for example, by up regulating the expression ofRGMc to decrease the permeability of the BBB or by down regulating theexpression of RGMa to increase the permeability of the BBB.

Multiple sclerosis is generally characterized as an immune-mediateddisease in which infiltrating immune cells damage myelin resulting in aloss of mobility and morbidity. However, in a subset of patients withprimary progressive multiple sclerosis (PPMS), neurodegeneration occursin the absence of immune-involvement. Treatments that can promoteregeneration of myelin and neuronal survival would be of value intreating PPMS and a number of other disorders associated withneurodegeneration. The Examples evidence that soluble RGMc can promoteremyelination in vitro and in vivo and therefore can be an effectivetreatment option for patients with PPMS and other neurodegenerativedisorders.

As used herein, the term “treatment” refers to administering acomposition of the invention to effect an alteration or improvement of adisease or condition. Prevention, amelioration, and/or treatment mayrequire administration of multiple doses at regular intervals, or priorto onset of the disease or condition to alter the course of the diseaseor condition. Moreover, a single agent may be used in a singleindividual for each of prevention, amelioration, and treatment of acondition or disease sequentially, or concurrently.

“Modulate”, as used herein, refers to a stimulatory or inhibitory effecton the intracellular process of interest relative to the level oractivity of such a process in the absence of a treatment as describedherein.

As used herein, “pharmaceutically acceptable carrier or excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, fillersand the like that are physiologically compatible. Examples ofpharmaceutically acceptable carriers include one or more of water,saline, phosphate buffered saline, dextrose, glycerol, ethanol and thelike, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the pharmacological agent.

As used herein, “therapeutically effective amount” refers to an amounteffective, at dosages and for a particular period of time necessary, toachieve the desired therapeutic result. A therapeutically effectiveamount of the pharmacological agent may vary according to factors suchas the disease state, age, sex, and weight of the individual, and theability of the pharmacological agent to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the pharmacological agent are outweighedby the therapeutically beneficial effects.

The pharmaceutical compositions as described herein may be administeredin a number of ways and, in one embodiment, the mode of administrationis not particularly restricted and, for example, administration may beby inhalation or in the form of ophthalmic or otic compositions;enteral, such as orally; or parenteral, including e.g. subcutaenous,intravenous, intra-arterial or intra-muscular. In one embodiment,administration is intravenous.

Various embodiments are disclosed including:

-   1. A method of decreasing the permeability of the blood brain    barrier in a subject comprising administering to the subject a    therapeutically effective amount of Repulsive Guidance Molecule C    (RGMc), soluble RGMc (sRGMc) or a functional fragment, variant or    mimic thereof.-   2. A method of stabilizing or restoring the blood brain barrier in a    subject comprising administering to the subject a therapeutically    effective amount of RGMc, sRGMc or a functional fragment, variant or    mimic thereof.-   3. A method of decreasing the permeability of the blood brain    barrier in a subject comprising administering to the subject a    therapeutically effective amount of an agent that inhibits Repulsive    Guidance Molecule A (RGMa).-   4. A method of stabilizing or restoring the blood brain barrier in a    subject comprising administering to the subject a therapeutically    effective amount of an agent that inhibits RGMa.-   5. The method of embodiment 3 or 4, wherein the agent inhibits    binding between RGMa and Neogenin or promotes binding between RGMc    and Neogenin.-   6. The method of embodiment 5, wherein the agent is an antibody, a    peptide agent, a cholesterol-lowering agent, or any combination    thereof.-   7. The method of embodiment 6, wherein the peptide agent is a RGMc    peptide, a Neogenin peptide, a Noggin peptide, fragment thereof,    variant thereof, or any combination thereof.-   8. The method of embodiment 3 or 4, wherein the agent is a    functional fragment of RGMc of at least 50 amino acids in length    that interacts with Neogenin.-   9. The method of embodiment 8, wherein the functional fragment of    RGMc is at least 50 amino acids in length and has a sequence    comprising an amino acid sequence found in SEQ ID NO: 1 or 3.-   10. The method of embodiment 7, wherein the peptide is sRGMc.-   11. The method of any one of embodiments 1 to 10, wherein the method    prevents or reduces immune cell infiltration into the central    nervous system (CNS).-   12. The method of any one of embodiments 1 to 11 for treating a    disease associated with disruption of the blood brain barrier.-   13. The method of any one of embodiments 1 to 12 for treating a    disease or condition, wherein the disease or condition is multiple    sclerosis (MS), ischemia (stroke), spinal cord injury, Alzheimer's    disease, Parkinson's disease, brain cancer, epilepsy, depression, or    an ocular condition, including glaucoma or retinitis pigmentosa    (RP).-   14. The method of embodiment 13, wherein the disease or condition is    MS.-   15. A method for disrupting blood brain barrier integrity in a    subject, comprising administering to the subject a therapeutically    effective amount of an agent that inhibits RGMc.-   16. A method for disrupting blood brain barrier integrity in a    subject, comprising administering to the subject a therapeutically    effective amount of an agent, wherein the agent is RGMa, sRGMa, a    functional fragment, variant or mimic thereof or stimulates RGMa.-   17. A method for increasing the permeability of the blood brain    barrier of a subject to a molecule present in the blood stream of    the subject comprising co-administering to said subject an effective    amount of an agent that inhibits RGMc.-   18. A method for increasing the permeability of the blood brain    barrier of a subject to a molecule present in the blood stream of    the subject comprising co-administering to said subject a    therapeutically effective amount of an agent, wherein the agent is    RGMa, sRGMa, a functional fragment, variant or mimic thereof or    stimulates RGMa.-   19. The method of any one of embodiments 15 to 18, wherein the    molecule and the agent are administered concomitantly.-   20. The method of any one of embodiments 15 to 18, wherein the agent    is administered prior to the molecule.-   21. The method of embodiment 20, wherein the agent is administered    about one day, more than one day, within about 23, 22, 21, 20, 19,    18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or    within one hour prior to administration of the molecule.-   22. The method of any one of embodiments 15 to 21 further comprising    administering a therapeutically effective amount of RGMc, sRGMc, or    a functional fragment, variant or mimic thereof to the subject after    administration of the molecule to decrease the permeability of the    blood brain barrier.-   23. The method of embodiment 22, wherein the therapeutically    effective amount of RGMc, sRGMc, or a functional fragment, variant    or mimic thereof is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9,    10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours    or is administered more than one day after administration of the    molecule.-   24. The method of any one of embodiments 15 to 22 further comprising    administering a therapeutically effective amount of an agent that    inhibits RGMa to the subject after administration of the molecule to    decrease the permeability of the blood brain barrier.-   25. The method of embodiment 24, wherein the therapeutically    effective amount of the agent that inhibits RGMa is administered    within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,    18, 19, 20, 21, 22, 23 or 24 hours or is administered more than one    day after administration of the molecule.-   26. The method of embodiment 24 or 25, wherein the agent that    inhibits RGMa inhibits binding between RGMa and Neogenin or promotes    binding between RGMc and Neogenin.-   27. The method of embodiment 26, wherein the agent is an antibody, a    peptide agent, a cholesterol-lowering agent, or any combination    thereof.-   28. The method of embodiment 27, wherein the peptide agent is an    RGMc peptide, a Neogenin peptide, a Noggin peptide, fragment    thereof, variant thereof, or any combination thereof.-   29. The method of any one of embodiments 15 to 28, wherein the    molecule is an imaging agent.-   30. The method of any one of embodiments 15 to 28, wherein the    molecule is a pharmaceutical.-   31. The method of embodiment 30, wherein the pharmaceutical is an    anaesthetic, antipsychotic, antidepressant, an antiemetic, or an    anticonvulsant.-   32. The method of embodiment 30, wherein the pharmaceutical is for    the treatment of a disease or condition selected from MS, ischemia    (stroke), spinal cord injury, Alzheimer's disease, epilepsy,    depression, or an ocular condition, including glaucoma or RP, or a    movement disorder, including Parkinson's disease.-   33. The method of embodiment 30, wherein the pharmaceutical is an    anti-cancer drug.-   34. The method of any one of embodiments 1 to 33 wherein the subject    is human.-   35. A method of determining the prognosis of a patient suffering    from a disease or condition associated with disruption of the blood    brain barrier comprising:-   determining the level of sRGMc and sRGMa in a patient sample;-   comparing the level of sRGMc and sRGMa in the patient sample to    average levels in a population sample of patients suffering from    said disease or condition, wherein a level of sRGMc below the    average and a level of sRGMa above the population sample average is    predictive of a higher than average severity of the disease or    condition.-   36. The method of embodiment 35 wherein the disease or condition is    MS, ischemia (stroke), spinal cord injury, Alzheimer's disease,    Parkinson's disease, brain cancer, epilepsy, depression, or an    ocular condition, including glaucoma or RP.-   37. The method of embodiment 35 or 36 wherein the sample is a blood    sample.-   38. The method of any one of embodiments 35 to 37 further comprising    treating the patient by a method of any one of embodiments 1 to 14.-   39. Use of a therapeutically effective amount of RGMc, sRGMc or a    functional fragment, variant or mimic thereof for decreasing the    permeability of the blood brain barrier in a subject.-   40. Use of a therapeutically effective amount of RGMc, sRGMc or a    functional fragment, variant or mimic thereof for stabilizing or    restoring the blood brain barrier in a subject.-   41. Use of a therapeutically effective amount of an agent that    inhibits RGMa for decreasing the permeability of the blood brain    barrier in a subject.-   42. Use of a therapeutically effective amount of an agent that    inhibits RGMa for stabilizing or restoring the blood brain barrier    in a subject.-   43. The use of embodiment 41 or 42, wherein the agent inhibits    binding between RGMa and Neogenin.-   44. The use of embodiment 43, wherein the agent is an antibody, a    peptide agent, a cholesterol-lowering agent, or any combination    thereof.-   45. The use of embodiment 44, wherein the peptide agent is a RGMc    peptide, a Neogenin peptide, a Noggin peptide, fragment thereof,    variant thereof, or any combination thereof.-   46. The use of embodiment 41 or 42, wherein the agent is a    functional fragment of RGMc of at least 50 amino acids in length    that interacts with Neogenin.-   47. The use of embodiment 46, wherein the functional fragment of    RGMc is at least 50 amino acids in length and has a sequence    comprising an amino acid sequence found in SEQ ID NO: 1 or 3.-   48. The use of embodiment 45, wherein the peptide is sRGMc.-   49. The use of any one of embodiments 39 to 48, wherein the use    prevents or reduces immune cell infiltration into the CNS.-   50. The use of any one of embodiments 39 to 49 for treating a    disease associated with disruption of the blood brain barrier.-   51. The use of any one of embodiments 39 to 50 for treating a    disease or condition, wherein the disease or condition is MS,    ischemia (stroke), spinal cord injury, Alzheimer's disease,    Parkinson's disease, brain cancer, epilepsy, depression, or an    ocular condition, including glaucoma or RP.-   52. The use of embodiment 51, wherein the disease or condition is    MS.-   53. Use of a therapeutically effective amount of an agent that    inhibits RGMc for disrupting blood brain barrier integrity in a    subject.-   54. Use of a therapeutically effective amount of an agent, wherein    the agent is RGMa, sRGMa, a functional fragment, variant or mimic    thereof or stimulates RGMa, for disrupting blood brain barrier    integrity in a subject.-   55. Use of a therapeutically effective amount of an agent that    inhibits RGMc for increasing the permeability of the blood brain    barrier of a subject to a molecule present in the blood stream of    the subject.-   56. Use of a therapeutically effective amount of an agent, wherein    the agent is RGMa, sRGMa, a functional fragment, variant or mimic    thereof or stimulates RGMa, for increasing the permeability of the    blood brain barrier of a subject to a molecule present in the blood    stream of the subject.-   57. The use of any one of embodiments 53 to 56, wherein the molecule    and the agent are administered concomitantly.-   58. The use of any one of embodiments 53 to 56, wherein the agent is    administered prior to the molecule.-   59. The use of embodiment 58, wherein the agent is administered    about one day, more than one day, within about 23, 22, 21, 20, 19,    18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or    within one hour prior to administration of the molecule.-   60. The use of any one of embodiments 53 to 59 further comprising    administering a therapeutically effective amount of RGMc, sRGMc, or    a functional fragment, variant or mimic thereof to the subject after    administration of the molecule to decrease the permeability of the    blood brain barrier.-   61. The use of embodiment 60, wherein the therapeutically effective    amount of RGMc, sRGMc, or a functional fragment, variant or mimic    thereof is administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,    12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours or is    administered more than one day after administration of the molecule.-   62. The use of any one of embodiments 53 to 59 further comprising    administering a therapeutically effective amount of an agent that    inhibits RGMa to the subject after administration of the molecule to    decrease the permeability of the blood brain barrier.-   63. The use of embodiment 62, wherein the therapeutically effective    amount of the agent that inhibits RGMa is administered within 1, 2,    3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,    22, 23 or 24 hours or is administered more than one day after    administration of the molecule.-   64. The use of embodiment 62 or 63, wherein the agent inhibits    binding between RGMa and Neogenin or promotes binding between RGMc    and Neogenin.-   65. The use of embodiment 64, wherein the agent is an antibody, a    peptide agent, a cholesterol-lowering agent, or any combination    thereof.-   66. The use of embodiment 65, wherein the peptide agent is an RGMc    peptide, a Neogenin peptide, a Noggin peptide, fragment thereof,    variant thereof, or any combination thereof.-   67. The use of any one of embodiments 53 to 66, wherein the molecule    is an imaging agent.-   68. The use of any one of embodiments 53 to 66, wherein the molecule    is a pharmaceutical.-   69. The use of embodiment 68, wherein the pharmaceutical is an    anaesthetic, antipsychotic, antidepressant, an antiemetic, or an    anticonvulsant.-   70. The use of embodiment 68, wherein the pharmaceutical is for the    treatment of a disease or condition selected from MS, ischemia    (stroke), spinal cord injury, Alzheimer's disease, epilepsy,    depression, or an ocular condition, including glaucoma or RP, or a    movement disorder, including Parkinson's disease.-   71. The use of embodiment 68, wherein the pharmaceutical is an    anti-cancer drug.-   72. The use of any one of embodiments 39 to 71 wherein the subject    is human.-   73. Use of RGMc, sRGMc, or a functional fragment, variant or mimic    thereof in the manufacture of a medicament for decreasing the    permeability of the blood brain barrier in a subject.-   74. Use of RGMc, sRGMc, or a functional fragment, variant or mimic    thereof in the manufacture of a medicament for stabilizing or    restoring the blood brain barrier in a subject.-   75. Use of an agent that inhibits RGMa in the manufacture of a    medicament for decreasing the permeability of the blood brain    barrier in a subject.-   76. Use of an agent that inhibits RGMa in the manufacture of a    medicament for stabilizing or restoring the blood brain barrier in a    subject.-   77. The use of embodiment 75 or 76, wherein the agent inhibits    binding between RGMa and Neogenin or promotes binding between RGMc    and Neogenin.-   78. The use of embodiment 77, wherein the agent is an antibody, a    peptide agent, a cholesterol-lowering agent, or any combination    thereof.-   79. The use of embodiment 78, wherein the peptide agent is a RGMc    peptide, a Neogenin peptide, a Noggin peptide, fragment thereof,    variant thereof, or any combination thereof.-   80. The use of embodiment 75 or 76, wherein the agent is a    functional fragment of RGMc of at least 50 amino acids in length    that interacts with Neogenin.-   81. The use of embodiment 80, wherein the functional fragment of    RGMc is at least 50 amino acids in length and has a sequence    comprising an amino acid sequence found in SEQ ID NO: 1 or 3.-   82. The use of embodiment 79, wherein the peptide is sRGMc.-   83. The use of any one of embodiments 73 to 82, wherein the use    prevents or reduces immune cell infiltration into the CNS.-   84. The use of any one of embodiments 73 to 83 for treating a    disease associated with disruption of the blood brain barrier.-   85. The use of any one of embodiments 73 to 84 for treating a    disease or condition, wherein the disease or condition is MS,    ischemia (stroke), spinal cord injury, Alzheimer's disease,    Parkinson's disease, brain cancer, epilepsy, depression, or an    ocular condition, including glaucoma or RP.-   86. The use of embodiment 85, wherein the disease or condition is    MS.-   87. Use of an agent that inhibits RGMc in the manufacture of a    medicament for disrupting blood brain barrier integrity in a    subject.-   88. Use of an agent, wherein the agent is RGMa, sRGMa, a functional    fragment, variant or mimic thereof or stimulates RGMa, in the    manufacture of a medicament for disrupting blood brain barrier    integrity in a subject.-   89. Use of an agent that inhibits RGMc in the manufacture of a    medicament for increasing the permeability of the blood brain    barrier of a subject to a molecule present in the blood stream of    the subject.-   90. Use of an agent, wherein the agent is RGMa, sRGMa, a functional    fragment, variant or mimic thereof or stimulates RGMa, in the    manufacture of a medicament for increasing the permeability of the    blood brain barrier of a subject to a molecule present in the blood    stream of the subject.-   91. The use of any one of embodiments 87 to 90, wherein the molecule    and the agent are for administration concomitantly.-   92. The use of any one of embodiments 87 to 90, wherein the agent is    for administration prior to the molecule.-   93. The use of embodiment 92, wherein the agent is for    administration about one day, more than one day, within about 23,    22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,    4, 3, 2, 1 or within one hour prior to administration of the    molecule.-   94. The use of any one of embodiments 87 to 93 in combination with a    therapeutically effective amount of RGMc, sRGMc, or a functional    fragment, variant or mimic thereof for administration to the subject    after administration of the molecule to decrease the permeability of    the blood brain barrier.-   95. The use of embodiment 94, wherein the therapeutically effective    amount of RGMc, sRGMc, or a functional fragment, variant or mimic    thereof is for administration within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,    11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours or is    for administration more than one day after administration of the    molecule.-   96. The use of any one of embodiments 87 to 93 in combination with a    therapeutically effective amount of an agent that inhibits RGMa for    administration to the subject after administration of the molecule    to decrease the permeability of the blood brain barrier.-   97. The use of embodiment 96, wherein the therapeutically effective    amount of the agent that inhibits RGMa is for administration within    1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,    20, 21, 22, 23 or 24 hours or is for administration more than one    day after administration of the molecule.-   98. The use of embodiment 96 or 97, wherein the agent inhibits    binding between RGMa and Neogenin or promotes binding between RGMc    and Neogenin.-   99. The use of embodiment 98, wherein the agent is an antibody, a    peptide agent, a cholesterol-lowering agent, or any combination    thereof.-   100. The use of embodiment 99, wherein the peptide agent is a RGMc    peptide, a Neogenin peptide, a Noggin peptide, fragment thereof,    variant thereof, or any combination thereof.-   101. The use of any one of embodiments 87 to 100, wherein the    molecule is an imaging agent.-   102. The use of any one of embodiments 87 or 100, wherein the    molecule is a pharmaceutical.-   103. The use of embodiment 102, wherein the pharmaceutical is an    anaesthetic, antipsychotic, antidepressant, an antiemetic, or an    anticonvulsant.-   104. The use of embodiment 102, wherein the pharmaceutical is for    the treatment of a disease or condition selected from MS, ischemia    (stroke), spinal cord injury, Alzheimer's disease, epilepsy,    depression, or an ocular condition, including glaucoma or RP or a    movement disorder, including Parkinson's disease.-   105. The use of embodiment 102, wherein the pharmaceutical is an    anti-cancer drug.-   106. The use of any one of embodiments 73 to 105 wherein the subject    is human.-   107. A pharmaceutical composition comprising RGMc, sRGMc or a    functional fragment, variant or mimic thereof and a pharmaceutically    acceptable carrier or excipient for decreasing the permeability of    the blood brain barrier in a subject.-   108. A pharmaceutical composition comprising RGMc, sRGMc or a    functional fragment, variant or mimic thereof and a pharmaceutically    acceptable carrier or excipient for stabilizing or restoring the    blood brain barrier in a subject.-   109. A pharmaceutical composition comprising an agent that inhibits    RGMa and a pharmaceutically acceptable carrier or excipient for    decreasing the permeability of the blood brain barrier in a subject.-   110. A pharmaceutical composition comprising an agent that inhibits    RGMa and a pharmaceutically acceptable carrier or excipient for    stabilizing or restoring the blood brain barrier in a subject.-   111. The pharmaceutical composition of embodiment 109 or 110,    wherein the agent inhibits binding between RGMa and Neogenin or    promotes binding between RGMc and Neogenin.-   112. The pharmaceutical composition of embodiment 111, wherein the    agent is an antibody, a peptide agent, a cholesterol-lowering agent,    or any combination thereof.-   113. The pharmaceutical composition of embodiment 112, wherein the    peptide agent is a RGMc peptide, a Neogenin peptide, a Noggin    peptide, fragment thereof, variant thereof, or any combination    thereof.-   114. The pharmaceutical composition of embodiment 109 or 110,    wherein the agent is a functional fragment of RGMc of at least 50    amino acids in length that interacts with Neogenin.-   115. The pharmaceutical composition of embodiment 114, wherein the    functional fragment of RGMc is at least 50 amino acids in length and    has a sequence comprising an amino acid sequence found in SEQ ID NO:    1 or 3.-   116. The pharmaceutical composition of embodiment 113, wherein the    peptide is sRGMc.-   117. The pharmaceutical composition of any one of embodiments 107 to    116, wherein the pharmaceutical composition prevents or reduces    immune cell infiltration into the CNS.-   118. The pharmaceutical composition of any one of embodiments 107 to    117 for treating a disease associated with disruption of the blood    brain barrier.-   119. The pharmaceutical composition of any one of embodiments 107 to    118 for treating a disease or condition, wherein the disease or    condition is MS, ischemia (stroke), spinal cord injury, Alzheimer's    disease, Parkinson's disease, brain cancer, epilepsy, depression, or    an ocular condition, including glaucoma or RP.-   120. The pharmaceutical composition of embodiment 119, wherein the    disease or condition is MS.-   121. A pharmaceutical composition comprising an agent that inhibits    RGMc and a pharmaceutically acceptable carrier or excipient for    disrupting blood brain barrier integrity in a subject.-   122. A pharmaceutical composition comprising an agent, wherein the    agent is RGMa, sRGMa, a functional fragment, variant or mimic    thereof or stimulates RGMa and a pharmaceutically acceptable carrier    or excipient, for disrupting blood brain barrier integrity in a    subject.-   123. A pharmaceutical composition comprising an agent that inhibits    RGMc and a pharmaceutically acceptable carrier or excipient, for    increasing the permeability of the blood brain barrier of a subject    to a molecule present in the blood stream of the subject.-   124. A pharmaceutical composition comprising an agent, wherein the    agent is RGMa, sRGMa, a functional fragment, variant or mimic    thereof or stimulates RGMa and a pharmaceutically acceptable carrier    or excipient for increasing the permeability of the blood brain    barrier of a subject to a molecule present in the blood stream of    the subject.-   125. The pharmaceutical composition of any one of embodiments 121 to    124, wherein the molecule and the agent are for administration    concomitantly.-   126. The pharmaceutical composition of any one of embodiments 121 to    124, wherein the agent is for administration prior to the molecule.-   127. The pharmaceutical composition of embodiment 126, wherein the    agent is for administration about one day, more than one day, within    about 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,    7, 6, 5, 4, 3, 2, 1 or within one hour prior to administration of    the molecule.-   128. The pharmaceutical composition of any one of embodiments 121 to    127 in combination with a further pharmaceutical composition    comprising a therapeutically effective amount of RGMc, sRGMc, or a    functional fragment, variant or mimic thereof and a pharmaceutically    acceptable carrier or excipient for administration to the subject    after administration of the molecule to decrease the permeability of    the blood brain barrier.-   129. The pharmaceutical composition of embodiment 128, wherein said    further pharmaceutical composition is for administration within 1,    2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,    21, 22, 23 or 24 hours or is for administration more than one day    after administration of the molecule.-   130. The pharmaceutical composition of any one of embodiments 121 to    127 in combination with a further pharmaceutical composition    comprising a therapeutically effective amount of an agent that    inhibits RGMa and a pharmaceutically acceptable carrier or excipient    for administration to the subject after administration of the    molecule to decrease the permeability of the blood brain barrier.-   131. The pharmaceutical composition of embodiment 130, wherein the    further pharmaceutical composition is for administration within 1,    2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,    21, 22, 23 or 24 hours or is for administration more than one day    after administration of the molecule.-   132. The pharmaceutical composition of embodiment 130 or 131,    wherein the agent inhibits binding between RGMa and Neogenin or    promotes binding between RGMc and Neogenin.-   133. The pharmaceutical composition of embodiment 132, wherein the    agent is an antibody, a peptide agent, a cholesterol-lowering agent,    or any combination thereof.-   134. The pharmaceutical composition of embodiment 133, wherein the    peptide agent is an RGMc peptide, a Neogenin peptide, a Noggin    peptide, fragment thereof, variant thereof, or any combination    thereof.-   135. The pharmaceutical composition of any one of embodiments 121 to    134, wherein the molecule is an imaging agent.-   136. The pharmaceutical composition of any one of embodiments 121 or    134, wherein the molecule is a pharmaceutical.-   137. The pharmaceutical composition of embodiment 136, wherein the    pharmaceutical is an anaesthetic, antipsychotic, antidepressant, an    antiemetic, or an anticonvulsant.-   138. The pharmaceutical composition of embodiment 136, wherein the    pharmaceutical is for the treatment of a disease or condition    selected from MS, ischemia (stroke), spinal cord injury, Alzheimer's    disease, epilepsy, depression, or an ocular condition, including    glaucoma or RP or a movement disorder, including Parkinson's    disease.-   139. The pharmaceutical composition of embodiment 136, wherein the    pharmaceutical is an anti-cancer drug.-   140. The pharmaceutical composition of any one of embodiments 107 to    139 wherein the subject is human.-   141. A method of promoting re-myelination in a subject comprising    administering to the subject a therapeutically effective amount of    Repulsive Guidance Molecule C (RGMc), soluble RGMc (sRGMc) or a    functional fragment, variant or mimic thereof.-   142. A method of preventing de-myelination in a subject comprising    administering to the subject a therapeutically effective amount of    Repulsive Guidance Molecule C (RGMc), soluble RGMc (sRGMc) or a    functional fragment, variant or mimic thereof.-   143. The method of embodiment 141 or embodiment 142, in which the    subject has primary progressive multiple sclerosis.-   144. Use of a therapeutically effective amount of RGMc, sRGMc or a    functional fragment, variant or mimic thereof for promoting    re-myelination in a subject.-   145. Use of a therapeutically effective amount of RGMc, sRGMc or a    functional fragment, variant or mimic thereof for preventing    de-myelination in a subject.-   146. The use of embodiment 144 or embodiment 145, in which the    subject has primary progressive multiple sclerosis.-   147. A pharmaceutical composition comprising RGMc, sRGMc or a    functional fragment, variant or mimic thereof and a pharmaceutically    acceptable carrier or excipient for promoting re-myelination in a    subject.-   148. A pharmaceutical composition comprising RGMc, sRGMc or a    functional fragment, variant or mimic thereof and a pharmaceutically    acceptable carrier or excipient for preventing de-myelination in a    subject.-   149. The composition of embodiment 147 or embodiment 148 for the    treatment of MS optionally primary progressive MS, ischemia    (stroke), spinal cord injury, Alzheimer's disease, Parkinson's    disease, brain cancer, epilepsy, depression, or an ocular condition    including optic neuritis, glaucoma or RP.

EXAMPLES Example 1

The documents referenced herein are incorporated by reference wherepermitted, however, it should be appreciated that any patent,publication, or other disclosure material, in whole or in part, that isincorporated by reference herein is incorporated only to the extent thatthe incorporated material does not conflict with definitions,statements, or other disclosure material set forth in this disclosure.As such, and to the extent necessary, the disclosure as explicitly setforth herein supersedes any conflicting material incorporated herein byreference.

Materials and Methods for Examples 2-10

Provided herein are in Example 1 are materials and methods that wereused in the experiments described below in Examples 2-10

Experimental Autoimmune Encephalomyelitis Mouse Model.

Six to eight week-old female C57BL/6 mice (Harlan laboratories) or129S-Hfe2^(tm1Nca)/J (stock. No. 017788, Jackson Laboratories) and theirwild type control 129S1/SvImJ (stock. No. 002448, Jackson Laboratories)were immunized by subcutaneous (s.c.) injection of 50 μg myelinoligodendrocyte glycoprotein (MOG) amino acids 35-55 (Sheldon Biotech,Montreal, QC) in incomplete Freund's adjuvant (Sigma) supplemented with1 mg of Mycobacterium tuberculosis (CFA) (Difco, Detroit, Mich.). 400 ngof pertussis toxin (List Biologicals) was administered intraperitoneally(i.p.) on days 0 and 2 post-immunization (FIG. 2). The animals werescored behaviorally using a well-established body condition score (BCS)using the following scale: 0, no paralysis; 1, loss of tail-tone reflex;2, loss of righting reflex; 3, complete hind limb paralysis; 4, forelimbweakness; 5, moribund or dead. Intermediate scores (0.5) were given toanimals that did not meet the upper scale of paralysis. A meancumulative score was obtained from two reading per day at 12 hintervals.

Administration of sRGMc and sRGMa.

To investigate the role of sRGMc, 20 mM of either purified sRGMc, sRGMa,or vehicle (PBS) were administered to mice induced with EAE byintra-venous (i.v.) injection at days 3, 6 and 9 or intra-peritoneallyevery 3d for the duration of the disease. To elucidate the physiologicalinteraction between sRGMc and sRGMa in the EAE model, a combination ofsRGMc (20 mM) in conjunction with 2 volumes of sRGMa (40 mM) wasadministered to EAE mice intra-peritoneally every 3d until the time ofsacrifice.

sRGMc cloning in the Psectag2B Vector.

Membrane-bound full-length mouse RGMc was used for further cloning. Thefull-length annotated mouse sequence including the GPI-anchor can befound using Uniprot AC/ID identifier Q7TQ32 on the Uniprot website. Thefull-length annotated sequence in Homo Sapiens including the GPI-anchorcan be found using Uniprot AC/ID identifier Q6ZVN8 on the Uniprotwebsite. To replicate endogenous soluble RGMc, present in serum, the GPIanchor sequence of RGMc was removed, generating a truncated form of RGMc(sRGMc). The resulting protein is targeted to the membrane using thePsectag2B signaling sequence and is released out of the cell, into themedium, as a soluble protein (human and mouse soluble proteins areprovided as SEQ ID NOs: 1 and 3, respectively, and their codingsequences as SEQ ID NOs: 2 and 4, respectively). sRGMC was generatedusing the following primers:

Forward primer: 5′ cttggtacccatcatcatcatcatcatcagtgcaagatcctccgctg 3′(SEQ ID NO: 5)

Reverse primer: 5′ gcgtctagacactcgagcgtcgagctgcccagctgtctgtc 3′ (SEQ IDNO: 6) Polymerase Chain Reaction (PCR) was performed using TAQpolymerase to generate an RGMc DNA fragment of 1175 base pairs in lengthcontaining a KpnI restriction site (5′ end) and an EcorI site (3′end)(New England Biolabs). The PCR product was then purified and ligatedinto a T-vector which was subsequently digested with the above-mentionedrestriction enzymes, inserted into a pSecTag2B expression vector(Invitrogen, Thermo Fisher Scientific) and the construct was sequencedto ensure the protein was in the correct frame. The pSecTag 2B vectorcontains a T7 promoter binding site and an Igk lead signal peptide forthe specific targeting of the inserted sRGMc sequence to the cellularmembrane.

Cell Culture.

Human Embryonic Kidney cells (HEK293) and murine brain-derivedEndothelial cells (b.end3) were cultured at 37° C. and 5% CO₂. Cellswere maintained in Dulbecco's Modified Eagle Medium (Sigma-Aldrich orATCC) containing 10% fetal bovine serum (FBS) (Gibco) and 1%penicillin/streptomycin (p/s) (Gibco).

sRGMc Expression.

sRGMc pSecTag2b plasmid was transiently transfected into HEK293 cellsusing polyethylenimine (PEI, Polysciences Inc.). Cells were transfectedwith 9 μg of DNA for 6 h at 37° C. and 5% CO₂, washed usingDulbecco-Phosphate Buffered Saline (D-PBS, Sigma-Aldrich) and media withreduced serum (OPTI-MEM, Gibco) was then added to the cells andincubated at 37° C. and 5% CO₂ for 48 h. The media was then harvested,centrifuged at 300 rcf for 5 min and supernatants were extracted andsRGMc was visualized by SDS-PAGE.

Neogenin Expression.

To verify Neogenin expression in endothelial cells, b.End3 cellmembranes were prepared as previously described. [Tassew N et al.(2014). Modifying Lipid Rafts Promotes Regeneration and FunctionalRecovery. Cell Rep 8:1146-1159] Briefly, cells were washed with chilledPBS, homogenized on ice using G27.5/G30 needles in homogenizing buffer(10 mM HEPES, 25 mM KCl, 5 mM MgCl₂, pH 7.3) with protease inhibitors.The homogenate was overlaid on a 50% and 5% sucrose gradient,centrifuged at 28,000 rpm in SW 60Ti rotor (Beckman) for 10 min at 4° C.The membranes were then extracted using a 21G needle and centrifuged at13,000 rpm for 10 min at 4° C. The supernatant was removed and themembrane pellets were re-suspended to the appropriate concentration inPBS. The membranes were then prepared for Western Blotting analysis.

sRGMc and sRGMa Proteins.

Mouse RGMa and RGMc were cloned with His tag replacing the GPI-anchor toallow secretion and subsequent purification of the proteins. HEK293cells (95% confluent) grown in antibiotic-free media were transfectedwith sRGMc or sRGMa using Lipofectamine 2000 (Invitrogen) according tomanufacturer's instructions. At 24 h post-transfection, cells werepassaged at a 1:3 ratio (37° C.) and at 48 h post-transfection,selection media (10% FBS, 1% p/s, 250 μg/mL Zeocin) was added to thecells. Each specific colony was then picked and expanded. The expressionof sRGMc and sRGMa was verified using western blot analysis. Media fromtransfected cells were collected 48 h later and purified on Ni-NTA beadsaccording to manufacturer's protocols (Qiagen). Proteins were dialyzedin PBS before being used in all assays.

Western Blot Analysis.

Cell lysates from mouse serum, cells, conditioned medium, andreduced-serum medium were incubated for 72 h and lysed using RIPA bufferand protease inhibitor cocktail. Western blots were performed aspreviously described. [Tassew N et al. (2014). Modifying Lipid RaftsPromotes Regeneration and Functional Recovery. Cell Rep 8:1146-1159] andprobed with primary antibodies (anti-RGMa AF-2459, R&D; anti-RGMc,AF-3636, R&D; anti-transferrin Sc-3-159, Santa Cruz; anti-NeogeninSc-15337, Santa Cruz). Donkey anti-mouse, goat anti-mouse, donkeyanti-goat secondary IRDye 800CW antibodies were used as appropriate(LI-COR BioSciences) for 1 h at room temperature. Coomassie staining wasperformed to ascertain equal total protein loading for each condition.

In Vitro Competition Binding Assay.

Using a 96-well microtiter plate (Corning), wells coated with 100 μl (10μg/mL) of Poly-L-Lysine (Sigma-Aldrich) incubated at 4° C. overnightwere then washed 3× with 100 μl of PBST (+0.02% Tween-20). His-taggedproteins (2.5 μg/mL of extracellular-Neogenin or sRGMa or sRGMc) werethen coated onto each well for 1 h at 37° C., washed 3× with 100 μlPBST, blocked with 300 μl of 3% BSA in PBST for 1 h at 37° C. and 50 μl(1.0 μg/mL) Alkaline Phosphatase (AP)-tagged proteins (extracellularNeogenin or RGMa or RGMc) in 1% BSA+PBST were added to each well andincubated at 37° C. for 1 h. Each well was washed 3× with 100 μl PBSTand equilibrated with AP developing buffer (100 mM NaHCO₃, 1 mM MgCl₂).The reaction was initiated using AP developing buffer supplemented withp-nitrophenyl phosphate (pNPP, Sigma-Aldrich) and then stopped followingcolor development by adding 50 μl (0.1M) NaOH. The absorbance of eachreaction was measured using a microplate autoreader (BioTek EL311AutoReader) at 405 nm.

Enzyme-Linked Immunoabsorbant Assay (ELISA).

Blood collected from the saphenous veins of mice induced with EAE at 0,5, 10, 18 and 30 days in Microvette® CB300 capillary tubes (Starstedt)was spun down at 2000 rcf for 10 min and stored at −80° C. sRGMa andsRGMc in the sera were detected using ELISA kits for sRGMa (MRGMAO, R&D)and sRGMc (MRGMCO, R&D) according to manufacturer's instructions.

To measure the expression of endogenous RGMc in mouse serum, prior toand 18 days post-EAE induction, blood was extracted from the saphenousvein of mice and allowed to coagulate for 2 h at room temperature. Theblood samples were then centrifuged at 2000 rcf for 20 min andsubsequently stored at −80° C. A 0.75 mm 4% stacking/12% SDS gel was runas previously described. The transferred nitrocellulose membrane wasblocked using 5% bovine serum albumin (BSA) (BioShop) in PBS. Blots werescanned and visualized using an Odyssey infrared imaging system. Proteinlevels were determined using densitometric analysis. RGMc protein levelswere then normalized to their corresponding transferrin protein levels(loading control).

Immune Cells Preparation.

Mice were euthanized at day 10 post-induction of EAE. Freshly isolatedspleens and draining caudal lymph nodes (LN) were pressed using asyringe plunger through a 70 μm cell strainers and washed twice using 2mL of 2% FBS in complete RPMI-1640 medium (Invitrogen) supplemented with243 Mercaptoethanol, L-glutamine, p/s, and non-essential amino acids.Single-cell suspensions were lysed with 7 mL of red blood cell (RBC)lysing buffer (Life Technologies) and centrifuged at 700 rcf for 5 mins.After washing three times with 2% RPMI-1640 medium, cells were countedand cultured as described below or were subjected to cell surfacestaining.

Flow Cytometry (FACS) and Intracellular Cytokine Analysis.

The expression of cell-surface markers and cytokine expression onsplenocytes and LN cells were characterized by flow cytometry. Foranalysis of naïve cells, 5×10⁵ RBC depleted splenocytes and LN cellswere plated in 96-well V-bottom plates (Starstedt) and blocked with 50μl of 1% mouse IgG in 2% FBS PBS supplemented with azide (PFN₃) for 20min at 4° C. Cells were then stained with the following primaryconjugated antibodies from BioLegend for 30 min on ice: CD5 (53-7.3),CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD19 (6D5), CD11b (M1/70),CD11c (N418), CD80 (16-10A1), CD86 (GL-1), CD44 (IM7), MHCII(M5/114.15.12), CD62L (MEL-14), ICAM-1/CD54 (3E2), IL-17A(TC11-18H10.41), IFN-γ (XMG1.2). All antibodies were re-suspended inPFN₃. When required, cells were washed twice with 100 μl of PFN₃ andstained with secondary SA-APC, SA-APC-Cy7, SA-PeCy7, or SA-Percp-Cy5.5for 30 min on ice. Cells were then washed twice with PFN₃ andre-suspended in a PFN₃ solution containing propidium iodide (PI) beforeproceeding to FACS analysis.

To examine intracellular cytokine production, 0.5×10⁶ RBC-depletedsplenocytes and LN cells were plated on 96-well flat bottom plates (BDBiosciences) in 10% FBS in complete RPMI-1640 in the presence of 0.5μg/mL phorbol 12-myrisate 13-acetate (PMA), 0.5 μg/mL Ionomycin, andGolgistop (BD Biosciences) for 4 h. Cells were subsequently washed inPBS and stained with Near-Infrared Live Dead (Life Technologies) asdescribed in the manufacturer's protocol. Cells were then washed twicein PFN₃ and stained for cell surface markers as described previously.Cells were then fixed with 100 μl of Cytofix/Cytoperm solution as permanufacturer's protocol. For intracellular staining, cells were thenstained with anti-IL-17A, and anti-IFN-γ for 30 min on ice, washedtwice, and re-suspended in PFN₃ before proceeding to FACS acquisition.Nonspecific background staining was determined by usingfluorochrome-matched isotype antibodies and fluorescence minus onecontrol. Samples were acquired in a BD LSRII or FACS Cantoll cytometer(BD Biosciences). For each experiment, at least 100,000 live events wereacquired and analyzed using Flowjo software (Tree Star Inc.).

MOG-Specific Cytokine Expression and Proliferation.

Mice were sacrificed on day 10 post-induction and single cellssuspension of splenocytes were harvested as described above. Cells(1×10⁶) were cultured in 10% FBS RPMI-1640 in the presence of 0 μg/mL,30 μg/mL, or 100 μg/mL of MOG peptide for 15 h. Cells were then treatedwith GolgiStop (BD Biosciences) and incubated an additional 3 h and weresubsequently stained.

Cytokine Analysis.

Briefly, 2×10⁶ splenocytes were re-suspended in 1 mL of 10% FBSRPMI-1640 medium and incubated in 24-well plates (BD Biosciences) with 0or 30 μg/mL MOG for 72 h (37° C., 5% CO₂). Plates were centrifuged at1800 rpm for 5 min and the media was then harvested and stored at −80 Cuntil analysis. Cytokine secretion of IL-17a, IL-6, TNF-α, and IFN-γwere assessed using the BD cytometric Bead Array (CBA) (BD Biosciences)according to the manufacturer's guidelines. Splenocyte proliferation wasdetermined using CellTrace™ CFSE Cell Proliferation Kit for flowcytometry (Life Technologies) according to manufacturer's instructions.Briefly, 1×10⁶ cells were suspended in warm PBS/0.1% BSA and stainedwith CFSE solution at a final concentration of 5 μM and incubated at 37°C. for 10 min. The staining was quenched using 5 volumes of ice-coldculture media, incubated for 5 min on ice, centrifuged at 700 rcf for 5min, washed twice using culture medium, and re-suspended in 10% FBScomplete RPMI-1640. Cells were then cultured in 0 μg/mL or 30 μg/mL ofMOG(35-55) peptide for 72 h at 37° C., 5% CO₂. Cells were thenharvested, washed, and stained with anti-CD3, -CD4, -CD5. Proliferationwas analyzed by flow cytometry following staining in the presence of PI.

Immunohistochemistry.

At 2 weeks post-induction of EAE, mice were sacrificed andtrans-cardially perfused with 20 mL PBS followed by 20 mL 4%paraformaldehyde in PBS (PFA) (Electron Microscopy Sciences). Spinalcords were isolated and post-fixed with 4% PFA overnight at 4° C.,washed 3× with PBS and soaked in 30% sucrose in PBS for 48 h. Tissueswere then embedded in Optimal Cutting Temperature (OCT) compound. Frozensections of spinal cords were cut at 10-30 μm with a cryostat, mountedon gelatinized slides, dried for 2 h and stored at −80° C. Sections werethen re-hydrated in PBS for 5 min and permeabilized with PBST (0.3%Triton X-100 in PBS) for 5 min. The tissues were then blocked (PBS, 0.3%Triton X-100, 3% FBS) for 1 h at room temperature and incubated with thefollowing primary antibodies overnight at 4° C. in a humidified chamber:anti-CD3 (145-2C11, BioLegend); CD11b (M1/70, BioLegend); B220 (RA3-6B2,BioLegend), DAPI (MMS-435P, Sigma), Fibrinogen (D9542, InnovativeResearch).

Histological Staining.

At day 18 post-EAE induction, mice were trans-cardially perfused with 20mL PBS followed by 20 mL 4% paraformaldehyde in PBS (PFA) (ElectronMicroscopy Sciences) and 30 μm spinal cord cryosections were stained.The slides were washed in PBS for 3 min, followed by a 30 s wash in 50%ethanol (EtOH) and incubated overnight in Luxol Fast Blue (LFB) at 60°C. The sections were washed in 95% EtOH for 5 min, distilled water for 5min prior to de-staining with LiCO2 for 10 min, and were then washed in70% EtOH for 30 s, 5 min in MilliQ H₂O, and sections were immersed inHarris Hematoxylin (Sigma-Aldrich) for 20 min and were rinsed in warmwater to remove excess stain. Samples were dehydrated in 95% EtOH,immersed in alcoholic eosin Y (Sigma-Aldrich) for 15 s, dehydrated twicein 95% EtOH for 5 min, 100% EtOH for 5 min and xylene for 5 min. Slideswere mounted with Permount (Fisher Scientific) and air-dried overnightat room temperature. Luxol Fast Blue (LFB) staining was quantified usingrelative staining intensity using Image J software. All intensities werenormalized to a sham mouse spinal cord.

Cellular infiltration was quantified using an inflammatory index: 0, noinflammation; 1, cellular infiltration only in the perivascular areasand meninges; 2, mild cellular infiltration in the parenchyma (<10cells); 3, moderate cellular infiltration in the parenchyma (10-49cells); 4, moderate cellular infiltration in the parenchyma (50-99cells); and 5, severe cellular infiltration in parenchyma (>100 cells).

In Vitro BBB Permeability.

b.End3 endothelial cells were used to generate an in vitro model of thehuman EC barrier lining the choroid plexus. Briefly, b.end3 cells wereplated on polyethylene-coated 0.4 μm pore size Boyden chambers (Corning)at a density of 2×10⁵ cells per well in DMEM, and were allowed to growfor 72 h to reach confluency (verified using crystal violet staining).After 72 h, cells were serum starved for 6 h and pre-treated for 4 hwith 10 μg/mL sRGMc, 10 μg/mL C3 Transferase or 10 μM Y27632. RGMa wasthen added to the wells for 18 h. The transwells were then transferredto a new plate containing 500 μl of HBSS and 200 μl 4-KDa Dextran-FITC(25 mg/ml; Sigma-Aldrich) was added to the upper chambers. Tracerdiffusion across the EC monolayer was assessed in 30 min intervals for aperiod of 1.5 h. FITC signal, which represented the amount ofextravasation through the monolayer was analyzed with using a microplatereader.

In Vivo BBB Permeability.

To establish the role of RGMc on BBB permeability, 6-8 week old C57Bl/6female mice induced with EAE and treated with sRGMc or vehicle (n=6animals per group) were anesthetized 18 days post induction andtrans-cardially perfused with 20 mL PBS followed by 20 mL 4%paraformaldehyde in PBS (PFA) (Electron Microscopy Sciences). Spinalcords were cryo-sectioned at 10 μm thickness and stored at −80° C. Thesections were stained for Fibrinogen (Innovative Research); CD31(Invitrogen); and DAPI (Sigma-Aldrich). All images were obtained using aBX61 confocal microscope (Olympus). CellSens software was used forquantification of cell area and pixel intensity. The area (number ofpixels) and fluorescent intensity (mean pixel intensity) of theextravasation markers were measured using CellSens software (Olympus).The relative extent of fluorescent extravasation and BBB disruption wasthen calculated by multiplying the area with the fluorescent intensity.

BBB Permeability.

Mice injected with BSA, RGMc, RGMa, or RGMa+RGMc. Widefield imaging wasthen carried out under an Olympus microscope (BX6 1WI) prior to andfollowing the i.v. injection of Texas red dye (100 μL, 5 mg/ml); imageswere captured using 100-300 ms exposure. Leakage of Texas red dye wasquantified by a blind experimenter measuring the number of leakagesites/mm². Recording was performed from 0 min to 35 min post-injection.

Adoptive Transfer.

EAE was passively induced in 6-8 week old C57Bl/6 mice were subsequentlytreated every 3 days with either RGMc or Vehicle (PBS) until sacrifice.Briefly, donor animals were immunized with 100 ug MOG(35-55) andsacrificed at day 10. Splenocytes were harvested and re-stimulated for 3days with 33 ug/mL MOG(35-55) in the presence of 20 ng/ml rmIL-23. Cellswere then labelled with CellVue Maroon and injected into recipientanimals treated with RGMc or vehicle as well as PTx on day 0 and day 2.Recipient animals were treated every 3 days and sacrificed on day 8 andspinal cords sectioned for immunofluorescence staining.

Statistical Analyses.

Data were analyzed using GraphPad Prism software. Student's t-tests andtwo-way ANOVA were used for direct comparisons between two groups. Forcomparisons between multiple groups, one-way ANOVA with Bonferronipost-hoc correction for multiple comparisons was utilized. Significancewas set at p<0.05 (*); p<0.01 (**); p<0.001 (***). Data are presented asmean and SEM.

Example 2 RGMc is Downregulated in EAE

To determine if levels of RGMc were affected by EAE, serum collectedfrom the saphenous vein of 3 EAE-induced mice at days 0, 5, 10, 18, and30 post-induction were analyzed for RGMc using a commercially availableRGMc ELISA kit and compared to control. Compared to control animals,RGMc levels were significantly reduced by day 5 post-induction andremained decreased for the duration of the disease course, reaching a4-fold decrease at 30 days post induction (FIG. 3A). This finding wasconfirmed by Western blot analysis (FIG. 3B). Thus, a significantrelationship was demonstrated between early development of EAE andreduced levels of circulating RGMc in EAE animals.

Example 3 RGMc Over-Expression Ameliorates the Clinical Severity ofEAE-Induced Mice

The observation that RGMc protein levels were significantly reducedfollowing induction of EAE led us to further investigate its role in theEAE model. Mice induced with EAE were treated with intravenousinjections of sRGMc at days 3, 6, and 9 post-induction. The sRGMcconstruct was generated as described in Materials and Methods; FIG. 4Ashows that sRGMc has a predicted molecular weight of ˜50 KDa innon-reducing conditions.

To ensure that over-expression of sRGMc was efficient, sera from 3RGMc-treated mice were collected and compared to 3 vehicle-treated miceat 24 h post-treatment by ELISA. Mice treated with sRGMc showed a 3-foldincrease in sRGMc levels compared to vehicle-treated animals. The micewere then observed and scored twice daily at 12 h intervals for 18 days.RGMc treatment resulted in a significant decrease in clinical severityof the disease (FIG. 4B), but these animals developed clinical symptomsby day 14 post-induction. Whether intra-peritoneal (i.p) injections ofRGMc could overcome the transient effects observed with intravenousadministration of this protein was explored. Indeed, ip injections ofRGMc significantly abrogated disease severity, decreased the incidenceof EAE onset (FIG. 4C), and delayed the onset of disease (FIG. 4D). Nextthe levels of RGMc in the sera of sham-treated animals and mice inducedwith EAE at day 10 as well as in EAE animals (day 10) that were injectedwith RGMc were analyzed. As shown in FIG. 4E, compared to sham-treatedanimals, RGMc levels were significantly reduced in EAE mice, which wererestored with RGMc injection. Thus, EAE reduced RGMc levels andover-expression of sRGMc in EAE-induced mice is beneficial in reducingthe incidence and the severity as well as delaying the onset of disease.

To assess the role of RGMc in the development of EAE, EAE was induced6-9 week old female mice with a selective homozygous deletion of RGMc(129S-Hfe2^(tm1Nca)/J). These mice recapitulate symptoms of JH, withearly onset iron overload as well as low hepcidin levels. Deletion ofRGMc was associated with a trend (p=0.06) towards an increase in diseaseseverity as well as percentage of incidence (FIG. 5). Thus, evidencingthat genetic neutralization of RGMc can result in an exacerbated diseasephenotype marked by an increased paralysis and a higher incidence of thedisease.

Example 4

sRGMa is Upregulated in EAE and is Crucial in the Development of theDisease.

Following the onset of EAE, membrane-bound RGMa is up-regulated on thesurface of immune cells, which increased their activation and subsequentextravasation to the CNS. [Muramatsu, R., Kubo, T., Mori, M., Nakamura,Y., Fujita, Y., Akutsu, T., Okuno, T., Taniguchi, J., Kumanogoh, A.,Yoshida, M., et al. (2011). RGMa modulates T cell responses and isinvolved in autoimmune encephalomyelitis. Nat. Med. 17, 488-494; KonigK, et al. (2012) The axonal guidance receptor neogenin promotes acuteinflammation. PloS one 7(3):e32145] RGMa has been shown to undergocomplex post-translational processing resulting in many biologicallyactive soluble fragments. [Tassew N G, Charish J, Seidah N G, & MonnierP P (2012) SKI-1 and Furin generate multiple RGMa fragments thatregulate axonal growth. Developmental Cell 22(2):391-402] Thus, thelevels of RGMa in the sera of mice induced with EAE at days 5, 10, 18and 30 post-EAE induction were analyzed. A dramatic increase (5-fold) inthe levels of RGMa in the sera of EAE-induced mice was observed, whichremained elevated for the entire duration of the disease (FIG. 6A).Thus, both sRGMa and sRGMc (FIG. 5F) are both present in mouse sera andpossess contrasting expression levels in EAE-induced mice.

To further analyze the role of sRGMc and sRGMa physiologically, whetherthe interaction of these proteins in mouse sera were crucial in thedevelopment of EAE was tested. Mice induced with EAE were treated witheither PBS (vehicle), sRGMc or with sRGMc in conjunction with sRGMa. Asshown in FIG. 6B, clinical severity was markedly reduced insRGMc-treated animals compared to control. Simultaneous treatment ofanimals with sRGMc with RGMa abolished the beneficial effects of RGMc(FIG. 6B) suggesting that RGMc and RGMa have contrasting effects on theclinical severity of EAE.

The possibility that RGMa and RGMc could interfere with each other'ssignaling by competing with binding to Neogenin was investigated.Pre-incubation of sRGMa with Ec-Neogenin-AP resulted in a 3-foldreduction binding intensity of RGMc to Neogenin when compared to BSAcontrol (FIG. 6C). The reversal experiment (FIG. 6D) showed a similarabrogation of binding intensity to Neogenin. Overall, these resultssuggest that sRGMa is able to interfere with sRGMc by interacting onnon-overlapping binding sites in Neogenin and vice-versa. Thus, thepresent inventors have shown for the first time that two molecules fromthe same protein family are able to compete and regulate each other'sbinding to their receptor and, furthermore, that sRGMa is critical inthe development of EAE.

Example 5

sRGMc has No Impact on Naïve Immune Cells.

Recent reports show that the RGMa-Neogenin signaling pathway is able tomodulate T cell priming and promote their activation by up-regulatingsmall GTPases such as Rap1. Activation of Rap1 increases the adhesionprofile of T cells as well as their infiltration within the CNS.[Muramatsu, R., Kubo, T., Mori, M., Nakamura, Y., Fujita, Y., Akutsu,T., Okuno, T., Taniguchi, J., Kumanogoh, A., Yoshida, M., et al. (2011).RGMa modulates T cell responses and is involved in autoimmuneencephalomyelitis. Nat. Med. 17, 488-494] Results showing that sRGMcinterferes with the interaction between sRGMa and Neogenin in vitroprompted investigation of its role in modulating the immune system. Toassess whether sRGMc alters the priming of immune cells, both thedistribution and activation of naïve immune cells of EAE-induced micewas analyzed. Splenocytes from day 10 RGMc-treated mice were analyzed byflow cytometry and compared to the vehicle-treated group. As shown inFIGS. 7 and 8, sRGMc treatment in EAE animals does not alter thedistribution of effector cells (CD3⁺, CD8⁺, and CD4⁺ T cells). Moreover,both the distributions and activation of dendritic cells (CD11c+) and Bcells (MHC II, CD80⁺/CD86⁺) were also unaltered. The infiltration ofleukocytes into the CNS is a key stage in the acute phase of EAE. As aresult, whether sRGMc could alter the ability of T cells to migrate intothe CNS was investigated. In order to migrate into the parenchyma,immune cells must overexpress a variety of cellular adhesion proteins.Here, the expression of adhesion markers (ICAM-1 and CD62L) in bothnaive splenocytes and lymph node cells was analyzed. As depicted in FIG.9, sRGMc treatment in EAE animals does not affect the expression ofadhesion markers on the surface of CD4⁺ and CD8⁺ T cells as well as Bcells.

Expression of RGMa on the surface of bone marrow-derived dendritic cellswas previously shown to prime and activate CD4⁺ T cells through itsinteraction with Neogenin and treatment with a polyclonal RGMa-antibodyreduced this activation demonstrated by reduced secretion of IL-17 andIFN-γ. [Muramatsu, R., Kubo, T., Mori, M., Nakamura, Y., Fujita, Y.,Akutsu, T., Okuno, T., Taniguchi, J., Kumanogoh, A., Yoshida, M., et al.(2011). RGMa modulates T cell responses and is involved in autoimmuneencephalomyelitis. Nat. Med. 17, 488-494] Here, the present inventorsshowed that sRGMc interferes with RGMa for its binding to Neogenin (FIG.6C). Consequently, whether treating EAE mice with sRGMc could alter theactivation profile of immune cells was explored. sRGMc was shown to haveno effect on the expression profile of IL-17 and IFN-γ secreted by CD4⁺T cells in EAE mice (FIG. 10). In the EAE model used, CD4⁺ T cells arespecifically primed towards MOG₍₃₅₋₅₅₎ peptide. Since the activationprofile of immune cells is dependent on their antigen recognition,whether presenting sRGMc-treated immune cells with MOG₍₃₅₋₅₅₎ peptidewould modulate the activation potential splenocytes when compared tocontrols was tested. C57/BL6 mice were immunized with MOG₍₃₅₋₅₅₎,followed by treatment with either sRGMc or vehicle and harvestedsplenocytes 10 days post induction. Splenocytes were then re-stimulatedwith MOG₍₃₅₋₅₅₎ peptide for 18 hrs and analyzed their cytokine profile.Again, no apparent effect was seen in RGMc-treated mice when compared tocontrol mice (FIG. 11). These findings were confirmed using an ELISA onmedia collected from splenocytes pulsed with MOG(35-55) for 3 days (FIG.12).

Example 6

sRGMc Alters Leukocyte Infiltration into the CNS

EAE is characterized by broad infiltration of immune cells within theCNS followed by de-myelination, both of which contribute to the clinicalseverity of the disease. [Gold R, Linington C, & Lassmann H (2006)Understanding pathogenesis and therapy of multiple sclerosis via animalmodels: 70 years of merits and culprits in experimental autoimmuneencephalomyelitis research. Brain: a Journal of Neurology 129(Pt8):1953-1971] Therefore whether sRGMc treatment alters leukocytepresence within the CNS of EAE-induced mice was explored. Thehistological and inflammatory profiles of cervical spinal cord sectionsisolated from vehicle- and RGMc-treated EAE animals were assessed.Strikingly, both the extent of cellular infiltrates and de-myelinationof sRGMc-treated animals were significantly reduced when compared tovehicle-treated animals (FIGS. 13A and B). Furthermore, the inflammatoryindex score was significantly lower in RGMc-treated animals (FIG. 13C).

To further characterize the profile of cellular infiltrates within thespinal cords of EAE-induced mice treated with RGMc, spinal cords werestained for CD3 (pan T cell marker), B220 (pan B cell marker) and CD11b(pan myeloid cell marker). In order to analyze the extravasation patternof each cell type within the spinal cord of EAE-induced mice, spinalcords were divided into 7 patterns of infiltration (data not shown). Asshown in FIG. 14, infiltration of CD3⁺, B220⁺, and Cd11b⁺ cells intocervical spinal cords harvested from EAE animals that received sRGMctreatment was significantly reduced, suggesting that sRGMc treatmentlessens the extent of leukocyte infiltration into the CNS in EAE-inducedmice. Furthermore, sRGMc treatment is associated with a preservedmyelination profile in contrast to vehicle-treated animals.

Example 7

sRGMc Reduces BBB Permeability in Endothelial Cells.

RGMa signaling is dependent on its interaction with Neogenin byactivating downstream cytoskeletal remodeling proteins, such as RhoA.[Conrad S, Genth H, Hofmann F, Just I, & Skutella T (2007) Neogenin-RGMasignaling at the growth cone is bone morphogenetic protein-independentand involves RhoA, ROCK, and PKC. The Journal of Biological Chemistry282(22):16423-16433]. Notably, RhoA signaling has extensively been shownto modulate the integrity of the blood-brain EC barrier. The BBBsynchronizes the homeostasis of the CNS, regulating the passage ofleukocytes into the parenchyma. Moreover, BBB disruption is a keyfeature in both EAE and MS, which leads to the subsequent infiltrationof immune cells within the CNS. [Persidsky Y, et al. (2006) Rho-mediatedregulation of tight junctions during monocyte migration across theblood-brain barrier in HIV-1 encephalitis (HIVE). Blood107(12):4770-4780] Moreover, the BBB could represent a site fortherapeutic target in CNS diseases. Based on observations that sRGMcinterferes with sRGMa binding to Neogenin and that sRGMc treatmentcurtails leukocyte infiltration into the CNS in EAE-induced mice, thepossibility sRGMc could restore BBB integrity by interfering with sRGMaactions was tested. To test this theory, a murine brain-derived cellline (b.end3) was obtained to replicate an in vitro model of theblood-brain EC barrier. Using both immunohistochemistry and western blotanalysis of b.end3 cells, it was shown that Neogenin is stronglyexpressed on the surface of these cells, as shown in FIG. 15A. Next therole of sRGMa on BBB permeability was assessed using a 4-KDa Dextranconjugated to a FITC fluorochrome (Dextran-FITC). b.end3 cells wereallowed to form a monolayer on Boyden chambers (FIGS. 14B and C) andwere subsequently treated with sRGMa, sRGMa blocking peptides for 4 hand extravasation of Dextran-FITC was assessed. Treatment of sRGMaincreased the extravasation of Dextran-FITC when compared tovehicle-treated wells. Treatment with either C3 Transferase (Rhoinhibitor) or Y27632 (Rho Kinase inhibitor) reduced the extravasation ofDextran-FITC (FIG. 15D). Because sRGMc reproduced the RhoA/RhoKinhibitor effect, the present inventors concluded that sRGMc by blockingRGMa-mediated Rho/RhoK activation diminishes blood-brain EC barrierpermeability, thus preventing infiltration of leukocytes in the CNS.

To confirm the role of sRGMc in maintaining blood-brain EC barrierintegrity in vivo, whether Neogenin was expressed in endothelial cellslining the BBB of mice was first examined. A sham mouse spinal cord wasstained with anti-CD31 to localize the presence of endothelial cells,DAPI to localize the nuclei of cell, as well as Neogenin. As shown inFIG. 16, Neogenin is widely expressed in the spinal cord andco-localizes with CD31 staining confirming that Neogenin is expressed onthe surface of endothelial cells in the mouse spinal cord. Next, thepresent inventors explored if sRGMc treatment alters the integrity ofthe BBB following the induction of EAE. Spinal cords isolated from RGMc-or vehicle-treated mice were stained with three markers: 1) Fibrinogen:a small plasma protein unable to cross the BBB under normal conditions2) CD31 and 3) DAPI. In sharp contrast to EAE animals treated withvehicle, animals treated with sRGMc showed a significant decrease infibrinogen extravasation (FIGS. 17A and B) indicative of a preserved BBBintegrity.

Example 8 RGMc Protects BBB Integrity

Next, the BBB permeability was assessed by injecting mice with BSA,RGMa, RGMa plus RGMc and subsequently followed by assessment of Texasdye leakage using widefield imaging as an indication of BBB integrity.RGMa significantly disrupted BBB integrity, which was suppressed by theRGMc addition (FIG. 18). Having shown that Neogenin is expressed byendothelial cells in human and murine tissues and because RGMa/cregulate BBB integrity, the present inventors considered whether RGMcregulates Claudin 5 levels, which is a tight junction protein criticalfor BBB maintenance. Thus, Claudin 5 expression following RGMa/ctreatments of murine-derived brain endothelial cells (b.End3) wasstudied. B.En3 cells were grown up to 80% confluency and were treatedwith overnight incubation with RGM proteins. Incubation with RGMc didaffect Claudin5 levels, but RGMa led to a 2-fold reduction in Claudin 5levels in these cells (FIG. 19). Strikingly, when RGMa and RGMc wereadded concurrently to cells, Claudin 5 levels were similar to PBSControls. Thus, RGMa induced down-regulation of Claudin 5 can beprevented by RGMc (FIG. 19). These data evidence a mechanism by whichRGMa and RGMc compete for binding to Neogenin, thereby regulatingClaudin5 levels and BBB integrity.

Example 9 RGMc Regulation of Cellular Infiltration

Having shown that RGMc levels regulate the extravasation of moleculesthrough the BBB, its role on cellular infiltration was evaluated. To doso, adaptive transfer using spleenocyte cells from MOG treated animalswas performed. After collection of splenocyte cells were re-stimulatedfor 3 days with MOG, stained with Cellvue Maroon and injected intorecipient mice treated with pertussis toxin. Following injection,animals received either PBS (Control) or RGMc every 3 days. Eight daysafter transfer animals were sacrificed and cellular infiltration wasmonitored using Cellvue-reporter. Examination of spinal cord sections ofPBS animals revealed an accumulation of fluorescent (Cellvue+)infiltrates that co-localized with T cell marker CD4 (FIG. 20). This wasin contrast with RGMc treated animals that did not show accumulation ofCellvue+ cells within the cord. Thus, treatment with RGMc prevented Tcell infiltration towards the CNS.

Example 10 Effects of RGMc, RGMa and Immunoglobulin Domain in Neogenin(4Ig) on BBB

The effects of RGMc, RGMa and the immunoglobulin domain in Neogenin (4Igpeptide) in modulating BBB integrity was explored by assessing Texas reddye leakage. To further assess the role of RGMc in modifying BBBintegrity, Texas dye leakage was also assessed in RGMc knockout (KO)mice. As observed in vitro, RGMa injection into mice disrupted BBBintegrity with significantly elevated leakage of Texas Red dye, whichwas ameliorated by RGMc or by blocking RGMa binding with Neognin (4Ig)(FIG. 21). Furthermore, similar experiments conducted in RGMc knockoutmice showed significantly less Texas Red dye leakage compared to wildtype animals (FIG. 21). Collectively, our data show that elevated RGMadisrupts BBB integrity, which can be restored by either RGMc or byblocking the interaction of RGMa with its receptor, Neogenin.

Example 11

Effects of sRGMc on Remyelination in Cerebellar Organotypic Cultures

To assess the ability of sRGMc to function as a remyelinating agent, itsfunction in in vitro cerebellar explant cultures was investigated.Briefly, cultures were demyelinated using lysolecithin, a detergent, andthen cultured in the presence of sRGMc for 14 days.

Myelination-demyelination-remyelination studies were performed in mousecerebellar organotypic cultures. The cerebella of postnatal 6 day oldpups were dissected and sectioned at 400 μm thickness using vibratome.Cerebellar sections were then plated on insert plates (Corning, WilkesBarre, Pa.) and allowed to attach. Media was replaced every 2-3 days andcultures were maintained for 7 days. To induce demyelination, 0.5 mg/mllysolecithin (1′-monoacyl-1-3-glycerylphosphorylcholine; Sigma) wasadded to the culture medium for 18 hr, after which it was removed andreplaced with fresh medium containing sRGMc (500 ng/ml) or controlprotein and the cultures maintained for 14 more days. Media was replacedevery 2-3 days. Demyelination-remyelination was assessed by stainingexplants with myelin basic protein antibody MBP (Cell Signaling).

Treatment with sRGMc (FIG. 22) was able to promote remyelination asassessed by staining with myelin basic protein. This finding evidencesthat sRGMc can be of therapeutic value in diseases impacted by loss ofmyelin, including primary progressive multiple sclerosis.

Example 12

Effects of sRGMc on Nerve Regeneration in an Optic Nerve Crush andRegeneration Model

Optic nerve crush experiments were performed on animals and recoveryassessed by staining for axonal fibres. The optic nerves of adult micewere crushed using fine self-closing forceps. sRGMc (1 μg/ml) or controlsolution was injected intraocularly at 3 and 10 days after injury.Animals were sacrificed at day 21 and optic nerves were dissected andfixed in 4% PFA. The optic nerves were sectioned longitudinally at 14microns and stained with GAP43 antibody to stain newly regeneratingaxons. The data showed that treatment with sRGMc, and not controlproteins, could promote the regeneration of axonal fibres at the site ofinjury, as was revealed upon staining with GAP43 (FIG. 23).

Example 13 Effects of RGMc in Stroke, as Modeled in a Middle CerebralArtery Occlusion (MCAO) Model

The possibility that RGMc may also restore BBB integrity followingstroke was also tested. Middle Cerebral Artery Occlusion (MCAO) wasperformed in rats, which were treated with a daily tail vein injectionof 70 μg of RGMc or PBS (control). Seven days following MCAO, animalsreceived injection of Evans blue to monitor BBB integrity (FIG. 24). Tomeasure BBB integrity, the amount of Evans Blue Dye/g of brain tissuewas measured. Animals that received RGMc showed a 3-fold reduction inEvans Blue levels in the brain compared to PBS-treated animals. Uponvisual inspection of the extent of Evans Blue dye for both sets ofbrains, it was seen that RGMc treatment strongly reduced Evans Blueextravasation into the CNS as compared to control (FIG. 24, A). Thiseffect was also quantitated: brains were homogenized and the amount ofEvans Blue quantified per gram of wet brain (FIG. 24, B). These data arecongruent with those from the EAE models, thus supporting our hypothesisthat RGMc can prevent BBB disruption in CNS injured animals.

In a separate experiment, whether RGMc treatment, which prevents BBBdisruption following stroke, could lead to functional recovery (FIG. 25)was studied. Rats were subjected to MCAO and treated with eithertail-vein injection of 70 μg of RGMc or control (PBS). Blindedbehavioral assessment of neurological deficit (Bederson test) wasperformed over a 7 day period, the results of which showed thattreatment with RGMc led to a significant (*p<0.05) improvement offunctional scores when compared to control. Our data suggest that RGMc,by preventing loss of BBB integrity and function, leads to restorationof neurological function following stroke.

1-74. (canceled)
 75. A method of decreasing the permeability of theblood brain barrier in a subject comprising administering to the subjecta therapeutically effective amount of an agent comprising animmunoglobulin domain of Neogenin.
 76. A method of stabilizing orrestoring the blood brain barrier in a subject comprising administeringto the subject a therapeutically effective amount of an agent comprisingan immunoglobulin domain of Neogenin.
 77. The method of claim 75 inwhich the agent comprises four immunoglobulin domains of Neogenin (4Ig).78. The method of claim 77 in which the agent comprises residues 1-417of GenBank accession AAI43272.
 79. The method of claim 77 in which theagent has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity tothe amino acid sequence comprising amino acids 1 to 383 of Accession No.AAC59662.
 80. The method of claim 75 for treating a disease or conditionassociated with disruption of the blood brain barrier.
 81. The method ofclaim 80 in which the disease is multiple sclerosis (MS), ischemia(stroke), spinal cord injury, Alzheimer's disease, Parkinson's disease,brain cancer, epilepsy, depression, or an ocular condition, includingglaucoma or retinitis pigmentosis (RP).
 82. The method of claim 81 inwhich the disease or condition is MS.
 83. The method of claim 82 inwhich the agent promotes re-myelination or prevents de-myelination inthe subject.
 84. The method of claim 80 in which the subject has primaryprogressive multiple sclerosis.
 85. The method of claim 80 in which themethod prevents or reduces immune cell infiltration into the centralnervous system (CNS).
 86. The method of claim 77 comprising, prior toadministration of the agent comprising 4Ig, administering an agent thatincreases the permeability of the blood brain barrier to a molecule inthe bloodstream of the subject.
 87. The method of claim 86 in which theagent that increases the permeability of the blood brain barrier isRepulsive Guidance Molecule A (RGMa), soluble RGMa (sRGMa), a functionalfragment, variant or mimic thereof or stimulates RGMa.
 88. The method ofclaim 86 in which administration of the agent comprises 4Ig isadministered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or is administered more thanone day after administration of the agent that increases thepermeability of the blood brain barrier.
 89. The method of claim 86 inwhich the molecule is a pharmaceutical or an imaging agent.
 90. Themethod of claim 89 in which the pharmaceutical or imaging agent isco-administered with or is administered after the agent that increasesthe permeability of the blood brain barrier.
 91. The method of claim 90in which the pharmaceutical is an anaesthetic, antipsychotic,antidepressant, antiemetic, anticonvulsant, or anti-cancer drug.
 92. Themethod of claim 89 in which the pharmaceutical is for treatment of adisease or condition selected from MS, ischemia (stroke), spinal cordinjury, Alzheimer's disease, epilepsy, depression, or an ocularcondition, including glaucoma or RP, or a movement disorder, includingParkinson's disease.
 93. The method of claim 75 in which the subject ishuman.
 94. The method of claim 75 in which the agent is a taggedpeptide.